Recipes
Recipes kdorfman Tue, 06/16/2009 - 20:04Antibodies
Antibodies kdorfman Tue, 10/02/2018 - 16:21For initial dilution of lyophilized antibody:
Dissolve to recommended concentration (per package insert) with half glycerol and half water.
Aliquot and freeze.
Label the tubes or the box with how much to dilute.
Primary Antibodies
Primary Antibodies kdorfman Tue, 10/02/2018 - 16:215-mC (rabbit)
5-mC (rabbit) kdorfman Fri, 11/05/2021 - 13:445-methylcytosine (D3S2Z)
Rabbit mAb
Binds to methylated cytosine
28692S Cell Signaling Technology
From the manufacturer: "Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1
Dilute 1:1600
Before Antibody treatment, treat fixed cells with 4N HCl for 15 minutes to denature DNA.
Neutralize with 2 washes with sodium borate pH 9, then PBS. Then start the PBS-Tw-Az rinses before incubating with the antibody.
Store at -20
Actin (mouse)
Actin (mouse) kdorfman Tue, 10/02/2018 - 18:50stock concentration = 22 ug/mL
working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 90 uL ~11 mg/mL
Add 216 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Actin (rabbit)
Actin (rabbit) kdorfman Tue, 10/02/2018 - 19:04Dilute 1:40 for use
Dilute 1:1 with glycerol
Aliquot 16 uL
Add 290 uL PBS BSA to make enough for 3 slides
Freezer 266A
Alkaline phosphatase (mouse)
Alkaline phosphatase (mouse) kdorfman Tue, 10/02/2018 - 18:51stock concentration = 38 ug/mL
diluted 1:1 in glycerol
50 uL aliquots of ~19ug/mL
Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides
266A freezer
Alpha-actinin (mouse)
Alpha-actinin (mouse) kdorfman Tue, 10/02/2018 - 17:46Binds actin to membrane at adherin junctions
Mouse monoclonal, Clone BM 75.2
IgM
Fix in formaldehyde (no MeOH or glutaraldehyde)
1:200
Use anti-mouse IgM FITC secondary
Cadherin (mouse)
Cadherin (mouse) kdorfman Tue, 10/02/2018 - 17:37"calcium-dependent adhesion"
cell adhesion molecule (CAM) important in formation of adherens junctions to bind cells with each other.
Class of type-1 transmembrane proteins.
Dependent on calcium (Ca2+) ions to function
small aliquot: 5 µL
large aliquot: 10 µL
Collagen II
Collagen II kdorfman Tue, 10/02/2018 - 18:59stock solution: 96 ug/mL
working solution: 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 20 uL ~48 mg/mL
Add 286 uL PBS-Tw-BSA to make enough for 3 slides
266A Freezer
Collagen IV
Collagen IV kdorfman Tue, 10/02/2018 - 19:00mouse anti collagen IV (basement membranes)
2 tubes as of spring 2018:
- stock concentration (8/7/08) = 30 ug/mL
- stock concentration (12/22/11) = 39 ug/mL
working concentration = 2 - 5 ug/mL
Each diluted with glycerol to a concentration of 18.5 ug/mL
Aliquots are 50 uL
Add 256 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Connexin (rabbit)
Connexin (rabbit) kdorfman Tue, 10/02/2018 - 19:07Freezer 266A
Anti-connexin-32 (265-279)
Dilute 1:600
Anti-Connexin-32 (106-124)
Dilute 1:400
Connexin (rat)
Connexin (rat) kdorfman Tue, 10/02/2018 - 19:17stock concentration = 28 ug/mL
working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 70 uL ~14 mg/mL
Add 236 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
DNMT1 (rabbit)
DNMT1 (rabbit) kdorfman Fri, 11/05/2021 - 13:39D63A6 XP(R)
Rabbit mAb
5032S Cell Signalling Technology
detects endogenous levels of total DNMT1 protein.
From the manufacturer: "Methylation of DNA at cytosine residues in mammalian cells is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting and development (1,2). Three families of mammalian DNA methyltransferases have been identified: DNMT1, DNMT2 and DNMT3 (1,2). DNMT1 is constitutively expressed in proliferating cells and functions as a maintenance methyltransferase, transferring proper methylation patterns to newly synthesized DNA during replication."
Dilute 1:100 for use
Golgi (mouse)
Golgi (mouse) kdorfman Tue, 10/02/2018 - 17:111 µL aliquots in tube. Store in freezer (-20C)
add 100 µL 1% BSA in PBS
Use just under 50 µL per coverslip (so there is enough for 2 per tube)
[edited 9/26/18 - used to say 1:200 dilution, but we had better results with 1:100]
Hec1 (mouse)
Hec1 (mouse) kdorfman Tue, 10/02/2018 - 16:23Novus Biologicals, recombinant human Hec1, monocolonal
fix in methanol
dilute 1:200
Histone H3 phospho S10
Histone H3 phospho S10 kdorfman Fri, 11/04/2022 - 20:46FINISH WORKING ON THIS!
Rabbit polyclonal
1 mg/mL, 100 ug, should be 100 uL in the package
1:2000
Storage:
- Dilute 1;10 glycerol
- 2 uL aliquots; for use, raise to 400 uL
- leave the rest with a label: dilute 1 uL in 200 uL PBS-Tw-Az-BSA
Integrin (rat)
Integrin (rat) kdorfman Tue, 10/02/2018 - 19:18rat anti-integrin-3
IgG2a
stock concentration = 27 ug/mL
working concentration = 2 - 5 ug/mL
Freezer 266A
LAMP1 (lysosomes) (mouse)
LAMP1 (lysosomes) (mouse) kdorfman Tue, 10/02/2018 - 16:22IgG
1 mg/mL
4C short term
Freeze 1 µL aliquots
dilute 1:100 - 1:750 in 0.1% BSA in PBS
rinse in PBS
Fix
- in 4% paraformaldehyde 15 min for endosomes
- in cold methanol for Golgi, 0.5% saponin 5 min
1% BSA in PBS 30 min
1o Ab stain (0.1% BSA) overnight at 4C
wash 30 min in PBS
2o Ab 1 h 37C
wash 30 min PBS
mount in DAPI
LAP2 (mouse)
LAP2 (mouse) kdorfman Tue, 10/02/2018 - 18:48Stains nuclear envelope
- fix in cold methanol
- 1:100
- BD transduction
- Mouse IgG
Lamin A/C
Lamin A/C kdorfman Fri, 11/04/2022 - 20:36mouse monocolonal (4C11)
7 ug/mL
Use at 1:100
(20 uL/class)
Storage:
- dilute 1:1 with glycerol,
- make 5 uL aliquots;
- add 245 uL PBS-Tw-Az-2% BSA
Lamin B1
Lamin B1 kdorfman Tue, 10/17/2023 - 16:30Rabbit anti-Lamin B1
Abcam AB16048
comes 100 µL at 1 mg/mL
Says to use at 0.1 µg/mL = 1:10,000 Drew's lab uses it at 1:1000
Make aliquots:
- 2 µL in a 2 mL tube
- label says
- 2 µL rabbit anti-lamin B1
- add 2 mL PBS-Tw-Azide
Aliquot and freeze - do not refreeze
Laminin (mouse)
Laminin (mouse) kdorfman Tue, 10/02/2018 - 19:01mouse anti-laminin gamma 1
stock concentration = 26 ug/mL
working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 80 uL ~12 mg/mL
Add 226 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Laminin (rabbit)
Laminin (rabbit) kdorfman Tue, 10/02/2018 - 19:08https://www.sigmaaldrich.com/catalog/product/sigma/l9393?lang=en®ion…
3 vials as of Spring 2018
dilute 1:25
266A freezer
Mucin (mouse)
Mucin (mouse) kdorfman Tue, 10/02/2018 - 19:03Monoclonal Anti-Mucin Gastric antibody produced in mouse
dilute 1:200 for use
dilute 1:1 in glycerol
Aliquot 4 uL; add 302 uL PBS BSA to make enough for 3 slides
Freezer 266A
Myosin (mouse)
Myosin (mouse) kdorfman Tue, 10/02/2018 - 18:46mouse anti-myosin light chain
stock solution: 37 ug/mL
working solution: 2 - 5 ug/mL
diluted 1:1 in glycerol
50 uL aliquots of ~18ug/mL
Add 256 uL PBS BSA to make enough at ~3ug/mL for 3 slides
Freezer 266A
Myosin (rabbit)
Myosin (rabbit) kdorfman Tue, 10/02/2018 - 19:14Neurofilament (rabbit)
Neurofilament (rabbit) kdorfman Tue, 10/02/2018 - 19:16Nuclear pore complex
Nuclear pore complex kdorfman Wed, 10/18/2023 - 19:16Anti-Nuclear Pore Complex Proteins antibody (Mab414)
100 µL, 1 mg/mL
Dilute 1:1000 to use
2 µL aliquots. Label says:
- add 198 µL PBS-Tw-Az-BSA
- dilute again 1:10 for use
Mouse monoclonal [Mab414] to nuclear pore complex proteins
Suitable for ICC or IF
Reacts with mouse, human, Saccharomyces cerevesiae
Isotype: IgG
Store at 4C short term
Aliquot and freeze for long term storage
Fix in 100% methanol -20C OR 4% PFA in PBS pH 7.4 10 min RT
PKC (rabbit)
PKC (rabbit) kdorfman Tue, 10/02/2018 - 20:44PKC alpha (c-20)
rabbit polyclonal Abs 1gG
DO NOT FREEZE
In refrigerator 36sA
dilute 1:50 to use
Santa Cruz Biotechnology sc-208
From Wikipedia: Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes.[5]
Protein kinase C-alpha (PKC-α) is a specific member of the protein kinase family. These enzymes are characterized by their ability to add a phosphate group to other proteins, thus changing their function. PKC-α has been widely studied in the tissues of many organisms including drosophila, xenopus, cow, dog, chicken, human, monkey, mouse, pig, and rabbit. Many studies are currently being conducted investigating the structure, function, and regulation of this enzyme. The most recent investigations concerning this enzyme include its general regulation, hepatic function, and cardiac function.
Phospho H3 (rabbit)
Phospho H3 (rabbit) kdorfman Mon, 08/19/2024 - 17:55For showing mitotic cells
Histone H3 phosphorylation on serine-10 is specific to mitosis and phosphorylated histone H3 (PHH3) proliferation markers (as counts defined per area or as indices defined per cell numbers) are increasingly being used to evaluate proliferation in various tumors.
Dilute 1:2000
Synaptic vesicle (mouse)
Synaptic vesicle (mouse) kdorfman Tue, 10/02/2018 - 18:57Synaptic vesicle glycoprotein 2A
Stock concentration = 36 ug/mL
Working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 60 uL ~18 mg/mL
Add 246 uL PBS-Tw-BSA to make enough for 3 slides
Freezer 266A
Troponin (mouse)
Troponin (mouse) kdorfman Tue, 10/02/2018 - 18:58DSHB TI-4-s
IgG (mouse)
Stock concentration = 43 ug/mL
Working concentration = 2 - 5 ug/mL
Diluted 1:1 with glycerol
Aliquots are 50 uL ~22 mg/mL
Add 256 uL PBS-Tw-BSA to make enough for 3 slides
266A Freezer
Tubulin alpha (rat)
Tubulin alpha (rat) kdorfman Tue, 10/02/2018 - 16:23Accurate Chem YSRTmcADg YSRTMCA77G
Clone YL 1/2
Antibody as purchased (2019) was diluted 1:1 with glycerol
As purchased, should be diluted 1:200; glycerol-diluted AB should be diluted 1:100 with PBS-Tw-Az-BSA
Each tube has 4 µL antibody
Add:
400 µL PBS-Tw-Az-BSA
=404 µL, enough for 8 reactions (50 µL per coverslip, 60 min 37C)
Incubate coverslip on 50 uL antibody 1 hr 37C in humid chamber
Tubulin gamma (mouse)
Tubulin gamma (mouse) kdorfman Tue, 10/02/2018 - 17:17binds to centrosomes
From Pat
Monoclonal, Sigma clone GTU-88
Fix in paraformaldehyde or cold methanol
Dilute 1:100
From Drew Sigma T6557-100UL
- Working concentration is 1:5000
- Frozen stock is diluted 1:1 with glycerol
- 5 uL aliquots (in half-mL tubes) should be diluted:
- 1:50 in the tube: add 245 uL PBS-Tw-BSA to make 1:100 stock
- 1:50 for use (1 uL 1:100 antibody + 49 uL PBS-Tw-Az-BSA per coverslip)
Drew's Protocol:
- Rinse in PBS
- Fix in paraformaldehyde 15 minutes
- Rinse with PBS-Triton
- Rinse with PBS-Tween
- Rinse with PBS-BSA 1 hour
- Dilute Ab 1:5000 in BSA
- Incubate with Ab 2hr RT
- Rinse with PBS 3x
- Incubate with secondary Ab (in BSA)
- Rinse with PBS 3x
- Mount
Test Protocol:
- coverslips already fixed in paraglut, stored in PBS-Tw-Az 1
- 1 uL Ab (as supplied by Sigma) into 99 uL PBS-BSA (save!)
- 1 uL 1:100 Ab into 499 uL PBS-BSA (save for the rest of the slides if it works!)
- 50 uL onto coverslip - 2 hr RT incubation
- rinse with PBS-Tw-Az
- 50 uL Goat anti mouse 488
-
Results: some background fluorescence. Add a blocking step:
rinse 30 min in 2% BSA
rinse 2x in PBS-Tw-Az
Didn't make much difference. centrosomes are small and bright ↩︎
Vinculin (mouse)
Vinculin (mouse) kdorfman Tue, 10/02/2018 - 18:44Membrane-cytoskeletal protein in focal adhesion plaques that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton, associated with cell-cell and cell-matrix junctions, where it is thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane.
Sigma V4505 monoclonal mouse
Fix in Formaldehyde
Dilute 1:100
ZO1 (Rabbit)
ZO1 (Rabbit) kdorfman Tue, 10/02/2018 - 16:24large tube has 10 µL antibody
small tube has 5 µL antibody
use at 1:100
362A freezer
fix in paraformaldehyde
Secondary antibodies
Secondary antibodies kdorfman Tue, 10/02/2018 - 19:19Goat anti-mouse IgM TRITC
Goat anti-mouse IgM TRITC kdorfman Tue, 10/02/2018 - 19:26Fisher OB102102 (Southern BioTech 1021-02)
Goat Anti-Mouse IgM (micron chain specific)-TRITC
1 mg/mL
1:100 - 1:400
1 µL aliquots. Add 100 - 400 µL 1% BSA
Use 50 µL per coverslip
Goat anti-mouse 488
Goat anti-mouse 488 kdorfman Tue, 10/02/2018 - 20:37Ex: 488 nm
Em: 499 nm
Stock(as purchased) = 2 mg/mL
Stock bottle has been diluted with glycerol: ~800 uL of 1 mg/mL
Working concentration = 1 ug/mL
Aliquots are 1 uL of 1 mg/mL
Add 611 uL to make enough for 6 slides, or up to 1 mL
Freezer 266A
Goat anti-mouse TRITC
Goat anti-mouse TRITC kdorfman Tue, 10/02/2018 - 19:23Anti-Mouse IgG (whole molecule) TRITC conjugate
Sigma T 5393
26 mg protein/mL
minimum working dilution = 1:64
1 µL aliquots. Add up to 100 µL 1% BSA
Alexa Fluor 568 (A11004) (red) 2mg/mL
working concentration ~10µg/mL
dilute 1:200
aliquots are 110 µL 20µg/mL
add
110 µL 2% BSA in PBS
to make 220 µL 10µg/mL
Use 50 µL per coverslip (there is enough for 4 per tube)
Goat anti-rabbit 546
Goat anti-rabbit 546 kdorfman Tue, 10/02/2018 - 20:39Stock concentration = 2 mg/mL
Working concentration = 4 ug/mL
Em = 575 nm
Ex = 560 nm
ALL OUT as of Spring 2018
Goat anti-rabbit FITC
Goat anti-rabbit FITC kdorfman Tue, 10/02/2018 - 20:42Goat anti-rabbit red
Goat anti-rabbit red kdorfman Tue, 10/02/2018 - 19:25Jackson Immuno 111-165-003
Cy3 Ex - 550 nm, em= 570 nm ("Red")
tubes : 2 µL. Add 398 µL PBS-Tw-Az to make 1:200
Use 50 µL per coverslip (there is enough for 4 per tube)
Goat anti-rat FITC
Goat anti-rat FITC kdorfman Tue, 10/02/2018 - 19:24Sigma F6258
Each tube has 3 µL antibody
Add
192 µL PBS-Tw-Az
195 µL 2% BSA
=390 µL, enough for 7 reactions (50 µL/coverslip 37C 30min)
Goat anti-rat TRITC
Goat anti-rat TRITC kdorfman Tue, 10/02/2018 - 19:31From Sigma AP136R EMB Millipore
ab peak = 550nm; emission peak = 570 nm
Bottle contains 2 mg. Add 1 mL H2O + 1 mL glycerol to make 1 mg/mL
aliquot 2 µL or 5 µL antibody, freeze
add 198 µL or 495 µL PBS-Tw-BSA
use 50 µL per coverslip, diluted to 1/100 (Try 37C 30 min)
Buffers
Buffers kdorfman Tue, 06/16/2009 - 17:51Here are some commonly made buffers for biochemistry and molecular biology
Citric acid buffer
Citric acid buffer kdorfman Tue, 11/16/2021 - 20:03Citric Acid – Na2HPO4 Buffer Preparation, pH 2.6–7.61
From Sigma Buffer Reference Center
Citric acid monohydrate, C6H8O7 • H2O, MW 210.14; 0.1 M contains 21.01 g/L.
Na2HPO4, MW 141.98; 0.2 M contains 28.40 g/L, or Na2HPO4 • 2H2O, MW 178.05; 0.2 M contains 35.61 g/L.
pH | x mL 0.1 M-citric acid | y mL 0.2-Na2HPO4 |
---|---|---|
2.6 | 89.10 | 10.90 |
2.8 | 84.15 | 15.85 |
3.0 | 79.45 | 20.55 |
3.2 | 75.30 | 24.70 |
3.4 | 71.50 | 28.50 |
3.6 | 67.80 | 32.20 |
3.8 | 64.50 | 35.50 |
4.0 | 61.45 | 38.55 |
4.2 | 58.60 | 41.40 |
4.4 | 55.90 | 44.10 |
4.6 | 53.25 | 46.75 |
4.8 | 50.70 | 49.30 |
5.0 | 48.50 | 51.50 |
5.2 | 46.40 | 53.60 |
5.4 | 44.25 | 55.75 |
5.6 | 42.00 | 58.00 |
5.8 | 39.55 | 60.45 |
6.0 | 36.85 | 63.15 |
6.2 | 33.90 | 66.10 |
6.4 | 30.75 | 69.25 |
6.6 | 27.25 | 72.75 |
6.8 | 22.75 | 77.25 |
7.0 | 17.65 | 82.35 |
7.2 | 13.05 | 86.95 |
7.4 | 9.15 | 90.85 |
7.6 | 6.35 | 93.65 |
For 2021 QBoC
Need pH 3,4,5,6,7
Start with
- Citric acid (100 mM) (borrowed from P-Chem)
- 2.65 g
- 125 mL H2O
- Sodium phosphate dibasic (200 mM)
- MW = 268.07
- 200 mM = 8.042 g
- 150 mL H2O
Then make
pH | x mL 0.1 M-citric acid | y mL 0.2-Na2HPO4 |
---|---|---|
3.0 | 39.725 | 10.275 |
4.0 | 30.75 | 19.275 |
5.0 | 24.25 | 25.75 |
6.0 | 18.425 | 31.575 |
7.0 | 8.825 | 41.175 |
Test pH of 1:10, 1:2
- pH remains constant when diluted to 20% with LB.
- So make 50 mL of each diluted to 40% with LB. Students will dilute it 1:1 with LB
- 20 mL buffer
- 30 mL water
- Filter steriize, aliquot for students
Cnidocyte washing buffer
Cnidocyte washing buffer kdorfman Thu, 10/10/2024 - 17:54Cnidocyte washing buffer
- 10 mM Tris
- 10 mM NaCl
- 10 mM EDTA
pH 7.6
Bring to pH 7.6, then final volume
Combine 400 ml of dH2O
0.6 g of Tris-HCl
0.29 g of NaCl
1.86 g of EDTA
Adjust the pH to 7.6 with 10 M NaOH.
Bring the volume to 500 ml with dH2O.
Store the buffer indefinitely at room temperature.
DEB
DEB kdorfman Mon, 12/19/2011 - 17:40DNA Extraction Buffer for Gene & Genome
- 100 mM NaCl
- 50 mM Tris, pH8
- 25 mM EDTA
- 1% SDS
Make from stock solutions
Diluent B
Diluent B kdorfman Mon, 09/26/2022 - 20:21Dilution buffer for NEB enzymes
Buffer composition:
Reagent | Final concentration |
---|---|
NaCl | 300 mM |
Tris-HCl | 10 mM |
DTT | 1 mM |
EDTA | 0.1 mM |
BSA | 500 ug/mL |
glycerol | 50% |
pH | 7.4 |
To make 50 mL for use, make 25 mL of 2x, then mix 1:1 with glycerol
25 mL 2x Diluent B
Reagent | c1 | c2 | v1 |
---|---|---|---|
NaCl | 5M | 0.3M | 3 mL |
Tris HCl | 1M | 0.01M | 0.5 mL |
DTT | 1 M | 1 mM | 0.05 mL |
EDTA | 500 mM | 0.1 mM | 0.01 mL |
plus BSA 25 mg
pH to 7.4, bring volume to 25, add glycerol to 50.
Filter sterilize
Edwards Extraction Buffer
Edwards Extraction Buffer kdorfman Tue, 03/22/2022 - 13:20For quickie DNA extraction from Arabidopsis
- 200 mM Tris pH 7.5
- 250 mM NaCl
- 25 mM EDTA
- 0.5% SDS
HBS (Ca-)
HBS (Ca-) kdorfman Tue, 12/27/2011 - 17:53Calcium-free HBS
Make from dry ingredients:
Compound | MW | mM | 1000 | 500 | 350 | 250 | 100 | mL |
---|---|---|---|---|---|---|---|---|
MgCl2(6H2O) | 203.3 | 0.493 | 0.1 | 0.05 | 0.035 | 0.025 | 0.01 | g |
KCl | 74.56 | 2.67 | 0.2 | 0.1 | 0.07 | 0.05 | 0.005 | g |
NaCl | 58.43 | 137.9 | 8 | 4 | 2.8 | 2 | 0.8 | g |
EGTA | 380.4 | 0.1 | 0.038 | 0.0192 | 0.0134 | 0.0095 | 0.00384 | g |
HEPES | 238.21 | 10 | 2.383 | 1.1915 | 0.858 | 0.596 | 0.238 | g |
raise pH to 7.3 with NaOH
1X HBS (ca-) from stock solutions
CHECK THIS
Ingredient | stock M | mM | 0.25 | 0.5 | 1 | 2 | L |
---|---|---|---|---|---|---|---|
MgCl.6H2O | 1 | 0.49 | 0.0001 | 0.0002 | 0.0005 | 0.0010 | mL |
KCl | 1 | 2.67 | 0.0007 | 0.0013 | 0.0027 | 0.0053 | mL |
NaCl | 5 | 137.90 | 0.0069 | 0.0138 | 0.0276 | 0.0552 | mL |
HEPES | 1 | 10 | 0. 025 | 0. 050 | 0. 100 | 0. 200 | mL |
pH 7.3 with NaOH
filter sterilize
HBS/Ca/Mg
HBS/Ca/Mg kdorfman Tue, 12/13/2011 - 20:32Ingredient1 | 1X (mM) | 10X (M) |
---|---|---|
CaCl2 | 0.9 | 0.009 |
MgCl2 | 0.493 | 0.00493 |
KCl | 2.67 | 0.0267 |
NaCl | 137.9 | 1.379 |
HEPES | 10 | 0.1 |
-
Concentrations from Lab 9, Bioimaging 2008, Dave Gross ↩︎
10X HBS (dry)
10X HBS (dry) kdorfman Fri, 08/09/2013 - 18:5510X HBS/Ca/Mg from dry ingredients
pH 7.3 with NaOH
Filter sterilize
10X HBS (stocks)
10X HBS (stocks) kdorfman Fri, 08/09/2013 - 18:561X HBS (stocks)
1X HBS (stocks) kdorfman Fri, 08/09/2013 - 20:22pH 7.3 with NaOH
Filter sterilize
HBSS
HBSS kdorfman Fri, 04/03/2020 - 14:57Hank's Balanced Salt Solution
stock bottle kept in tissue culture fridge
To make 1 Liter from dry ingredients
Components | MW | (mg/L) | mM |
---|---|---|---|
Calcium Chloride (CaCl2) (dihyd.) | 147 | 185 | 1.26 |
Magnesium Chloride (MgCl2-6H2O) | 203 | 100 | 0.493 |
Magnesium Sulfate (MgSO4-7H2O) | 246 | 100 | 0.407 |
Potassium Chloride (KCl) | 75 | 400 | 5.33 |
Potassium Phosphate monobasic (KH2PO4) | 136 | 60 | 0.441 |
Sodium Bicarbonate (NaHCO3) | 84 | 350 | 4.17 |
Sodium Chloride (NaCl) | 58 | 8000 | 137.93 |
Sodium Phosphate dibasic (Na2HPO4-6H20) | 268 | 90.6 | 0.338 |
D-Glucose (Dextrose) | 180 | 1000 | 5.56 |
Filter sterilize
To make from stock solutions:
Components | Ci (M) | cf (mM) | 1000 | 500 | 250 | mL |
---|---|---|---|---|---|---|
CaCl2 | 0.5 | 1.26 | 2.52 | 1.26 | 0.63 | mL |
MgCl2 | 1 | 0.493 | 0.493 | 0.247 | 0.108 | mL |
MgSO4 | 1 | 0.407 | 0.407 | 0.204 | 0.102 | mL |
KCl | 1 | 5.33 | 5.33 | 2.67 | 0.131 | mL |
KH2PO4 | 1 | 0.441 | 0.441 | 0.22 | 0.11 | mL |
NaHCO3 | 0.5 | 4.17 | 8.34 | 4.17 | 0.209 | mL |
NaCl | 5 | 137.93 | 27.59 | 13.79 | 6.9 | mL |
Na2HPO4 | 1 | 0.338 | 0.338 | 0.169 | 0.085 | mL |
Glucose | - | - | 1 | 0.5 | 0.25 | g |
Filter sterilize
HEPES
HEPES kdorfman Tue, 12/20/2011 - 14:16HEPES 1M
MW = 238.3
7.149 g / 30 mL
11.915 g / 50 mL
pH to 7.3 with NaOH pellets (~5 g/L)
Filter sterilize.
HotSHOT genomic DNA prep
HotSHOT genomic DNA prep kdorfman Tue, 06/21/2022 - 15:00For fin clip, tail snip, or ear punch
- Obtain tissue 2 mm
- tail snip
- ear punch
- fin clip
- Place tissue in 96 well plate
- Add 75 uL Alkaline Lysis Reagent for mouse, 50 uL for fish
- Heat to 95C 30 min (10 min to 1 hr)
- Cool to 4C
- Add 75 uL Neutralization Buffer for mouse, 50 uL for fish
- Use 3 uL (1-5) pre PCR reaction
Notes:
- DNA is suitable for PCR, not Southerns
- Heating longer than 30 min does not increase [DNA]
- pH of reagents does not need to be altered
- DNA yield is similar for tail snips and ear punches
- Too much tissue destroys PCR
- Store DNA at 4C or 20C
Alkaline Lysis Reagent
Alkaline Lysis Reagent kdorfman Tue, 06/21/2022 - 15:07Alkaline Lysis Reagent for HotSHOT DNA prep
Recipe from Mike Charles 10/15/03; mikchar@umich.edu; retrieved from Craig Albertson's lab, recalculated for1 M NaOH - 10 M will damage your plastic tube!!
Reagent | stock conc | final conc | amt |
---|---|---|---|
NaOH | 1 M | 25 mM | 1.25 mL |
EDTA | 0.5 M | 0.2 mM | 20 uL |
dI water | to final volume | 50 mL |
Calculator
pH =~12
Neutralization Buffer
Neutralization Buffer kdorfman Tue, 06/21/2022 - 15:09PBS
PBS kdorfman Tue, 06/16/2009 - 17:52Make 1X PBS from 10X
100 mL 10X + 900 mL H2O
10X PBS from Fisher
Fisher BP399-4 $51.07 as of 05/2020 ($34.45 January 2018!)
https://punchout.fishersci.com/shop/products/phosphate-buffered-saline-…
10X PBS from stock solutions
from Sigma ready-made 1X:
M | ingredient |
---|---|
0.01 | Na-K Phosphate |
0.138 | NaCl |
0.0027 | KCl |
pH to 7.3 with NaOH
sterilize
10X PBS from dry ingredients (Carrie's recipe)
pH to 7.3 with NaOH
sterilize
from stock solutions
from stock solutions kdorfman Wed, 11/13/2013 - 18:1310X PBS from stock solutions
from Sigma ready-made 1X:
M (1X) | M (10X) | ingredient |
---|---|---|
0.01 | 0.10 | Na-K Phosphate |
0.138 | 1.38 | NaCl |
0.0027 | 0.027 | KCl |
pH to 7.3 with NaOH
sterilize
OR
To match Sigma recipe:
Make 1M Na2HPO4 (base); 1 M KH2PO4 (acid)
Mix to pH 7.3
(should be ~38.25 mL Na2HPO4, 11.5 mL KH2PO4) from the Sigma Buffer Reference Center
pH | mL Na Phos dibasic | mL Na Phos monobasic |
---|---|---|
7.2 | 36.0 | 14.0 |
7.4 | 40.5 | 9.5 |
Then
PBS-Tw for Marine Bio
PBS-Tw for Marine Bio kdorfman Thu, 10/10/2024 - 17:380.1% Tween in PBS
PBS-Tw-Azide
PBS-Tw-Azide kdorfman Tue, 12/27/2011 - 18:34PBS with:
- 0.1% Tween 20
- 0.2 g/L Na Azide (=0.02 %)
per Liter:
- 100 mL 10x PBS
- 10 mL 10% Tween 20
- 2 mL 10% Na azide
PBX (for fish)
PBX (for fish) kdorfman Mon, 03/23/2020 - 13:570.5% Triton X in PBS
Plus water to final volume
PBX/BSA
PBX/BSA kdorfman Tue, 11/10/2020 - 18:44PBX with 3% BSA for fish
PIPES
PIPES kdorfman Thu, 01/19/2012 - 16:430.5 M pH 6.7
(for transformation buffer for genetics)
MW (disodium salt) = 346.32
173.16 g/L
17.316 g/ 100 mL
Mix in ~80% of final volume
pH to 6.7 with 5M KOH
Filter sterilize
Aliquot
Freeze
Potassium phosphate
Potassium phosphate kdorfman Sat, 01/14/2012 - 04:581 M Potassium phosphate buffer
To make 100 mL of 1M potassium phosphate:
pH | mL 1M K2HPO4 | mL 1M KH2PO4 |
---|---|---|
5.8 | 8.5 | 91.5 |
6.0 | 13.2 | 86.8 |
6.2 | 19.2 | 80.8 |
6.4 | 27.8 | 72.2 |
6.6 | 38.1 | 61.9 |
6.8 | 49.7 | 50.3 |
7.0 | 61.5 | 38.5 |
7.2 | 71.7 | 28.3 |
7.4 | 80.2 | 19.8 |
7.6 | 86.6 | 13.4 |
7.8 | 90.8 | 9.2 |
8.0 | 94.0 | 6.0 |
from http://ivaan.com/protocols/151.html
Check the pH with the meter.
Add more K2 to raise the pH, or K1 to lower it.
Dilute as necessary to achieve the desired concentration.
Sodium Phosphate
Sodium Phosphate kdorfman Mon, 12/12/2011 - 19:20To make 100 mL 1M sodium phosphate at a given pH:
pH | 1 M Na2HPO4 | 1 M NaH2PO4 |
---|---|---|
8.0 | 93.2 ml | 6.8 ml |
7.8 | 89.6 ml | 10.4 ml |
7.6 | 84.5 ml | 15.5 ml |
7.4 | 77.4 ml | 22.6 ml |
7.2 | 68.4 ml | 31.6 ml |
7.0 | 57.7 ml | 42.3 ml |
6.8 | 46.3 ml | 53.7 ml |
6.6 | 35.2 ml | 64.8 ml |
6.4 | 25.5 ml | 74.5 ml |
6.2 | 17.8 ml | 82.2 ml |
6.0 | 12.0 ml | 88.0 ml |
5.8 | 7.9 ml | 92.1 ml |
Dilute to the desired concentration.
Sodium acetate buffer
Sodium acetate buffer kdorfman Tue, 11/16/2021 - 19:53From Sigma Buffer Reference Center
Sodium Acetate – Acetic Acid Buffer PREPARATION | pH 3.7–5.61 Sodium acetate trihydrate, CH3COONa • 3H2O MW 136.09; 0.2 M contains 27.22 g/L.
x mL 0.2 M-NaOAc and y mL 0.2 M-HOAc mixed
pH @ 18 °C | x mL 0.2 M-NaOAc | y mL 0.2 M-HOAc |
---|---|---|
3.7 | 10.0 | 90.0 |
3.8 | 12.0 | 88.0 |
4.0 | 18.0 | 82.0 |
4.2 | 26.5 | 73.5 |
4.4 | 37.0 | 63.0 |
4.6 | 49.0 | 51.0 |
4.8 | 59.0 | 41.0 |
5.0 | 70.0 | 30.0 |
5.2 | 79.0 | 21.0 |
5.4 | 86.0 | 14.0 |
5.6 | 91.0 | 9.0 |
Sodium borate buffer
Sodium borate buffer kdorfman Fri, 11/05/2021 - 14:10(to neutralize acid wash during anti-5mC staining)
1L 1M
g | ingredient |
---|---|
61.83 g | Boric acid (H3BO3) |
10 g | NaOH |
water to 1L
100 mL 100mM
g | ingredient |
---|---|
0.6183 g | Boric acid (H3BO3) |
0.1 g | NaOH |
water to 100 mL
T10E1
T10E1 kdorfman Mon, 12/19/2011 - 17:54Tris 10 mM, EDTA 1 mM, pH 8
Make from stock solutions
ingredient | cf | ci | 25 | 50 | mL | ||
---|---|---|---|---|---|---|---|
Tris pH 8 | 10 | mM | 1000 | mM | 0.25 | 0.5 | mL |
EDTA | 1 | mM | 500 | mM | 0.05 | 0.1 | mL |
T10E5
T10E5 kdorfman Mon, 12/19/2011 - 18:25Tris 10 mM, EDTA 5 mM, pH 8
Make from stock solutions
ingredient | cf | ci | 25 | 50 | mL | ||
---|---|---|---|---|---|---|---|
Tris pH 8 | 10 | mM | 1000 | mM | 0.25 | 0.5 | mL |
EDTA | 5 | mM | 500 | mM | 0.25 | 0.5 | mL |
TAE
TAE kdorfman Tue, 06/16/2009 - 17:52Tris-Acetate-EDTA buffer for agarose gel electrophoresis
1X TAE
1X TAE kdorfman Tue, 06/16/2009 - 19:02plus water to final volume
50X TAE stock
50X TAE stock kdorfman Tue, 06/16/2009 - 19:01Buy from Fisher TAE 50x, pH 8.3, 4L, part #BP13324
from Maniotis
pH to 8 with acetic acid or NaOH
should this be 8.4?
Ingredient | cf (mM) | 1000 | 750 | 600 | 500 | mL |
---|---|---|---|---|---|---|
Tris | 2000 | 242 | 181.5 | 145.2 | 121 | g |
Acetic acid | 1000 | 57.1 | 42.9 | 34.3 | 28.6 | mL |
EDTA 0.5 M | 50 | 100 | 75 | 60 | 50 | mL |
OR | ||||||
EDTA | 50 | 18.62 | 13.965 | 11.172 | 9.31 | g |
pH to 8 with acetic acid or NaOH
Maniotis says to autoclave, but the salt concentration is so high that nothing will grow in it if you don't.
TBE 10X stock
TBE 10X stock kdorfman Thu, 08/29/2024 - 22:14BE Buffer 10x Stock Recipe:
- 108 g tris base.
- 55 g boric acid.
- 900 ml double-distilled H2O.
- 40 ml 0.5 M EDTA solution (pH 8.0)
Taq dilution buffer
Taq dilution buffer kdorfman Fri, 05/31/2013 - 17:21To dilute Takara Ex-Taq
Store in freezer. Won’t freeze solid. Should keep forever.
50 μL Tween 20
50 μL Nonidet P-40 (anionic detergent = NP-40)
10 µL 1M DTT
1 mL KCl (1M)
200 μL Tris-Cl pH 8 (1M)
2 μL 0.5M EDTA pH 8
5 mL glycerol
4.2 mL ddH2O
bring to final volume, filter sterilize in BSC, freeze
Culture Media
Culture Media kdorfman Fri, 04/27/2018 - 15:16Bacterial media
Bacterial media kdorfman Thu, 01/17/2013 - 18:53Freezing bacteria
Freezing bacteria kdorfman Sat, 12/05/2015 - 18:44To Freeze:
Pick a new colony, grow overnight in LB
inoculate LB from the overnight culture
Put 0.15 mL glycerol into sterile cryovial.
Add 0.85 mL mid-log culture (OD =~0.4). Pipette up and down to mix thoroughly
Freeze. Store in -80 if possible.
To thaw:
Scrape surface of frozen stock with sterile stick
Streak on agar plate
LB Broth
LB Broth kdorfman Fri, 09/25/2020 - 17:54LB broth from mix
LB broth from mix kdorfman Tue, 05/28/2013 - 17:48LB Broth Miller Luria-Bertani
Fisher DF0446-17-3 500 g $40.61.
25 g/L
LB from scratch
LB from scratch kdorfman Fri, 09/25/2020 - 17:38LB (Miller) from scratch
LB agar
LB agar kdorfman Thu, 01/17/2013 - 18:55LB agar granules
LB agar granules kdorfman Tue, 06/25/2019 - 20:01Make with granulated LB (Miller) agar, Fisher BP9724
LB agar powder
LB agar powder kdorfman Tue, 06/25/2019 - 20:00Make with LB (Miller) Agar powder, Difco 244510
LB broth + agar
LB broth + agar kdorfman Tue, 06/25/2019 - 20:0025 g LB + 15 g agar /liter
LB antibiotic
LB antibiotic kdorfman Wed, 10/01/2014 - 16:34Table of antibiotic stability in poured agar
line | antibiotic |
---|---|
black | Carbenicillin (ampicillin) |
green | IPTG |
LB-sugars
LB-sugars kdorfman Wed, 10/01/2014 - 16:19LB + sugars for Lac-operon work
sugar (or analog) | MW |
---|---|
mono saccharide | 180.2 |
disaccharide | 342.3 |
IPTG | 238.3 |
glucose, galactose, or fructose
Lactose is not very soluble in cold water.. Boil water, then set it to stir on a stir plate. Gradually tap in the lactose powder.
lactose, maltose, sucrose
IPTG
Filter sterilize
Add 50 µg/mL Kanamycin as indicated here.
MacConkey agar
MacConkey agar kdorfman Thu, 01/17/2013 - 19:41MacConkey Agar
- Fisher 212122 2 kg (Difco)
- Krackeler 10-211387 500g (via Sigma)
- HiMedia MacConkey agar, granulated GM081 (Fisher NC1876763, VWR 95020-204)
to distinguish Lac+ and Lac- bacterial strains.
50 g/L
(If you are going to make more than one bottle, turn on 65C waterbath in tissue culture room)
Turn on the stir-plate heat to 105C.
Withhold 50 mL of water to rinse the mouth of the bottle.
Heat the rest of the water to boiling in the bottle in the microwave.
Measure out the agar mix while the water is heating
Add the stir bar (CAREFULLY!) to the bottle of hot water
Put the bottle on the stir-plate
Add the agar mix to the bottle
Use the reserved water to rinse the granules stuck in the neck down to the liquid.
Stir and heat until granules are completely dissolved.
Cap loosely, wrap with foil, and autoclave.
(If you make more than one bottle, the extras go in the water bath till you are ready to pour)
leave stir bar in
put bottles on stir plate near sterile hood until handle-able
If you accidentally buy
MacConkey + bile+ CV
MacConkey + bile+ CV kdorfman Mon, 01/08/2024 - 16:00If you accidentally buy MacConkey without
- bile salts (1.5 g/L)
- Crystal violet (0.001g/L)
- NaCl (5 g/L)
Min med for GFP cells
Min med for GFP cells kdorfman Tue, 10/23/2018 - 15:53From https://wahoo.cns.umass.edu/node/857
For SfGFP cells, try M9 + 0.2% glycerol + 0.01 mM FeSO4
Mueller Hinton agar
Mueller Hinton agar kdorfman Thu, 01/17/2013 - 20:0590922 Mueller Hinton Broth 2 from Sigma
22 grams per liter, consisting of:
Casein acid hydrolysate 17.5
Beef extract 3.0
Starch 1.5
Final pH 7.3 +/- 0.2 at 25°C
P-Glo Bacterial media
P-Glo Bacterial media kdorfman Mon, 08/26/2019 - 22:26Make 60 mm plates
plain LB plates
LB/Amp (or Carb) plates (1 black stripe)
LB/Amp/Ara plates (1 black, 1 red stripe)
2019 - for QBoC:
- transformed pglo plasmid (1660405EDU) from the BioRad kit into
- DH5 alpha E. coli (Invitrogen 18265-017) from the -80
- Lyophilized cells (BioRad 1660408EDU) did not arrive in time
Used the Invitrogen protocol.
Plated on:
- LB
- LB amp
- LB amp ara
Saw gfp colonies on the arabinose plates. Collected, grew up in LB amp, froze in 15% glycerol.
LB-Amp-Ara
LB-Amp-Ara kdorfman Mon, 08/26/2019 - 22:27LB with Ampicilin or Carbenicilin and arabinose
Arabinose stock 200mg/mL in water, sterilized by filtration (=100 X)
Final concentration = 2 mg/mL
- Arabinose MW = 150.13
- 2 mg/mL = 2 g/L x 1 mol/150.13 g = 0.013M = 13 mM
Carbenicilin stock is 1000X
minimal medium (glycerol)
minimal medium (glycerol) bcrcstaff Wed, 11/14/2018 - 17:34For motility strains
Cell culture media
Cell culture media kdorfman Tue, 10/11/2011 - 19:24FBS aliquots
FBS aliquots kdorfman Tue, 10/11/2011 - 20:15FBS comes in 500 mL bottles.
Aliquot:
volume (mL) | # aliquots | for |
---|---|---|
50 | 3 | 500 mL DMEM or non-CO2 medium |
37.5 | 4 | 500 mL F10-Ham's |
25 | 4 | 250 mL DMEM or non-CO2 medium |
18.75 | 4 | 250 mL F10 Ham's |
6.25 | 4 | to make freezing media |
2013: Try
- Fisherbrand™ Research Grade Fetal Bovine Serum
- 03-600-511
- 500 mL for $133
Krackeler 45-F0926-500ML $145
Carrie's brand: Atlanta Biologicals Premium S11150 ~$300
Also use Krackeler 12103C ~$314
2019 Genessee GenClone FetalPURE™ Cat #: 25-525 $247.45
Antibiotic/Antimycotic
Antibiotic/Antimycotic kdorfman Tue, 10/11/2011 - 20:21Anti-Anti comes in 100 mL bottles.
Aliquot:
volume (mL) | # aliquots | for |
---|---|---|
10 | 1 | 1 L |
5 | 12 | 500 mL |
2.5 | 12 | 250 mL |
Keep frozen until ready to use.
Fisher SV30079.01, $20
DC5 (DMEM for canine cancer)
DC5 (DMEM for canine cancer) kdorfman Thu, 08/17/2023 - 20:00from Kathleen Arcaro
bring to final volume, filter sterilize in BSC, refrigerate
DMEM
DMEM kdorfman Tue, 10/11/2011 - 19:30Medium for 3t3 fibroblasts and B16 cells
bring to final volume, filter sterilize in BSC, refrigerate
- if using DMEM without pyruvate, add 1 mL Na Pyruvate (100mM) per 100 mL medium
Ingredient | supplier | cat # | 1 L | 500 mL | 250 mL | |
---|---|---|---|---|---|---|
DMEM | 13.4 | 6.7 | 3.35 | g | ||
NaHCO3 | 3.7 | 1.85 | 0.925 | g | ||
HEPES | 1.3 | 0.65 | 0.325 | g | ||
FBS | Krackeler | 12103C | 100 | 50 | 25 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix DMEM, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.5
Adjust pH to 7.2 with HCl (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.
F10-Ham's
F10-Ham's kdorfman Tue, 10/11/2011 - 19:49for all LLCPk cell lines
Thaw FBS and anti-anti first!
bring to final volume, filter sterilize in BSC, refrigerate
Ingredient | supplier | cat # | 1 L | 500 mL | 250 mL | |
---|---|---|---|---|---|---|
F10 (Ham's) | Sigma | N6635 | 4.9 | 2.45 | 1.225 | g |
Optimem | Invitrogen | 22600-050 | 6.8 | 3.4 | 1.7 | g |
NaHCO3 | 1.8 | 0.9 | 0.45 | g | ||
HEPES | 0.66 | 0.33 | 0.165 | g | ||
FBS | Krackeler | 12103C | 75 | 37.5 | 18.75 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix F10, Optimem, NaHCO3, HEPES in about 70% of the final volume of dH2O.
Initial pH ~7.1
Adjust pH to 7.2 (it will rise to 7.3 in the CO2 incubator).
Add appropriate aliquot of FBS and antibiotic/antimycotic.
Bring to final volume.
Filter sterilize in the tissue culture hood.
Refrigerate
For serum free, replace serum with distilled water
Freezing media
Freezing media kdorfman Tue, 10/11/2011 - 20:42Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.
F10 Hams for freezing (initial serum concentration = 0.075)
DMEM for freezing (initial serum concentration = 0.1)
Filter sterilize
McCoy's 5A
McCoy's 5A kdorfman Tue, 09/19/2023 - 15:19McCoy's 5A (modified) (Fisher 16600-082)
For HCT116 cells (a human colorectal carcinoma cell line initiated from an adult male. The cells are adherent with an epithelial morphology. Following implantation into immunocompromised mice, the cells form primary tumors and distant metastases. In vitro, HCT116 cells grow with a doubling time of about 18 hours.)
Drew's recipe:
10% FBS, 1% Pen-strep
- 500 mL McCoy
- 55 mL FBS
- 5.5 mL pen-strep
OR:
- remove 55 mL from the 500 mL McCoy's from Fisher
- add 50 mL FBS
- add 5 mL pen strep (e.g., MP 1670246)
- resterilize
Filter sterilize
Non-CO2 Media
Non-CO2 Media kdorfman Tue, 12/27/2011 - 18:37Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammal…
FluoroBrite™ DMEM Media
serum-free
serum-free kdorfman Wed, 10/19/2011 - 15:07Non-CO2 serum-free medium
For working with live cells at the microscope
bring to final volume, filter sterilize in BSC, refrigerate
Ingredient | supplier | cat # | 1000 | 500 | 250 | mL |
---|---|---|---|---|---|---|
MEM | Sigma | M3024-1L | 13.4 | 6.7 | 3.35 | g |
HEPES | Acros | 172571000 | 1.3 | 0.65 | 0.325 | g |
Na pyruvate 100 mM | Thermo | SH30239.01 | 10 | 5 | 2.5 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix MEM and HEPES in about 70% of the final volume of dH2O.
Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.
with serum
with serum kdorfman Tue, 10/11/2011 - 20:00Non-CO2 Medium
For working with live cells at the microscope, when serum is needed for normal division.
bring to final volume, filter sterilize in BSC, refrigerate
Ingredient | supplier | cat # | 1 L | 500 mL | 250 mL | |
---|---|---|---|---|---|---|
MEM | 13.4 | 6.7 | 3.35 | g | ||
HEPES | 1.3 | 0.65 | 0.325 | g | ||
Na pyruvate 100 mM | 10 | 5 | 2.5 | mL | ||
FBS | Krackeler | 12103C | 100 | 50 | 25 | mL |
anti/anti | Fisher | SV30079.01 | 10 | 5 | 2.5 | mL |
Mix MEM and HEPES in about 70% of the final volume of dH2O.
Initial pH ~6.3
Adjust pH to 7.3
Add appropriate aliquot of FBS, antibiotic/antimycotic, Na pyruvate
Bring to final volume.
Filter sterilize in the tissue culture hood.
Store in the refrigerator.
Trypsin
Trypsin kdorfman Tue, 01/19/2016 - 18:58Try Fisher 12-605-010
Stable at room temp!
Gibco™ TrypLE Express Enzyme (1X), Phenol Red
Animal origin-free, recombinant enzyme
$20.10 -for 100 mL
E2 medium (Zebrafish)
E2 medium (Zebrafish) kdorfman Sun, 01/13/2019 - 21:48Use the attached file while this set of pages is under construction
E2 Embryo Medium final Working Solution: 0.5X E2
To make 20 L, start with 19 L RO water, and add
ml | component |
---|---|
100 | 100X E2A |
20 | 500X E2B |
20 | 500X E2C |
10 | 0.1% MeBlue (optional) |
Then bring to final volume
0.1% Methylene Blue
0.1% Methylene Blue kdorfman Thu, 03/19/2020 - 14:531 gram of methylene blue per liter of RO water.
Shake well to dissolve.
Store at room temp.
0.5X E2A
0.5X E2A kdorfman Thu, 03/19/2020 - 14:54E2A (100x)
E2A (100x) kdorfman Thu, 03/19/2020 - 14:51FOLLOW THE RECIPE IN THE ATTACHED FILE (omitting methylene blue). THIS PAGE IS NOT DONE YET
100X E2:
1.5 M NaCl
50 mM KCl
100 mM MgSO4
15 mM KH2PO4
5 mM Na2HPO4
E2A Buffer Mix
E2A Buffer Mix kdorfman Thu, 03/19/2020 - 14:52E2B 500X
E2B 500X kdorfman Thu, 03/19/2020 - 14:52E2C 500X
E2C 500X kdorfman Thu, 03/19/2020 - 14:53Naegleria medium
Naegleria medium kdorfman Wed, 01/17/2018 - 14:34Medium 21
Medium 21 kdorfman Wed, 12/15/2021 - 21:44Mark with 2 red lines
Add 1 black line for carbenicillin (ampicillin)
Add 1 green line for IPTG
M21 from stocks
M21 from stocks kdorfman Wed, 12/15/2021 - 21:19Add 50 - 100 µg/mL ampicillin (carbenicillin) and 1 mM IPTG if needed
Medium 21 from dry
Medium 21 from dry kdorfman Tue, 12/14/2021 - 21:09If required, add IPTG 0.5mM (0.5 mL 1M per L medium) to 1 mM (1 mL 1M per L medium),
and ampicillin
NM from dry
NM from dry kdorfman Wed, 01/17/2018 - 14:34If required, add IPTG 0.5mM (0.5 mL 1M per L medium) to 1 mM (1 mL 1M per L medium),
and ampicillin
NM from stock solutions
NM from stock solutions kdorfman Wed, 01/17/2018 - 14:35DIW (pure deionized water) 780 ml
1M K2HPO4 (dibasic) 4 mL
1M KH2PO4 (monobasic) 16 mL
Difco Bacto Peptone (not Proteose Peptone) 3.2 g
Difco Bacto Agar 12.0 g
Mix by swirling (no need to dissolve). Autoclave 10 min. Cool to 45–50° in a water bath, and pour plates about ½ full.
Nematostella
Nematostella kdorfman Sat, 06/22/2024 - 18:18Anemones with Symbiodinium (symbiotic photosynthetic dynoflagellates) for Marine Bio
Nematostella culture instructions powerpoint
Culture in 12 - 15 psu (practical salinity units = g/L) ASW (artificial sea water), made with Instant Ocean Reef Crystals
Plant growth media
Plant growth media kdorfman Thu, 05/23/2013 - 16:0210 g agar per L
1/2 MS low Iron
1/2 MS low Iron kdorfman Tue, 02/15/2022 - 15:001/2 Murashige & Skoog, 1µM iron medium
MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM, 1/2 MS is 5µM
FeSO4 stock is 10 mM, which is 10,000x 1 µM
Fern Culture Media
Fern Culture Media kdorfman Thu, 01/11/2024 - 19:16Pre-made fern medium, Carolina 156780, sold in 60 mL or 400 mL
Carolina recommends 10 mL per plate. I use 9 mL
To get gametophytes in all stages, sow fern spores in 12 60 mm dishes 4 times:
- 1/15
- 1/22
- 1/29
- 2/2 or Friday 2/3
Fern Medium from scratch
Fern Medium from scratch kdorfman Fri, 01/12/2024 - 21:18To make one liter
800 mL water
10X macronutrients: 100mL
200X micronutrients: 5 mL
100X Chelated Iron solution: 10 mL
pH to 6 with NaOH
Bring final volume to 1 L
Add 10 g Bacto agar (others may not work)
Autoclave 15 min
10X fern macronutrients
10X fern macronutrients kdorfman Fri, 01/12/2024 - 21:24200X fern micronutrients
200X fern micronutrients kdorfman Fri, 01/12/2024 - 21:26Fern medium from powder
Fern medium from powder kdorfman Wed, 01/24/2024 - 14:20Mix contents of vial with 800 mL water
pH to 6 (NaOH)
Bring to 1 L
For single batch:
Add 10g Bacto Agar
Autoclave 45 min
For small batches:
Aliquot 167 mL into 6 200 mL media bottles
Add 1.67 g Bacto Agar to each bottle
Autoclave 30 min
LPGM
LPGM kdorfman Wed, 08/31/2016 - 20:24MS high salt
MS high salt kdorfman Mon, 02/03/2014 - 15:33150 too high - try 100mM for F 2015
150 mM NaCl
10 g agar/L
Make regular MS, then add 0.03 mL 5M NaCl per mL medium
100 mM NaCl
0.02 mL 5M NaCl per mL medium
MS iron-free
MS iron-free kdorfman Thu, 05/23/2013 - 17:46Murashige & Skoog 1µM iron medium
MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM
FeSO4 stock is 10 mM, which is 10,000x 1 µM
10 g agar/L
MS low iron
MS low iron kdorfman Thu, 05/23/2013 - 17:40Murashige & Skoog 1µM iron medium
MS 10x micronutrients is 100 µM FeSO4, so MS complete is 10 µM
FeSO4 stock is 10 mM, which is 10,000x 1 µM
MS medium
MS medium kdorfman Thu, 05/23/2013 - 16:021/2 MS
1/2 MS kdorfman Wed, 12/22/2021 - 20:25*Add the other salt mixtures to the water to prevent precipitation
**pH to 5.7 with 1M KOH (initial pH = ~3.66) (needs~21 drops or ~720 µL/L)
Autoclave with stir bar in flask or bottle
Stir until cool enough to handle
Pour 30 mL per plate (use the deep ones)
Pour 50 mL per square plate (20 plates per liter)
[1]: Sigma M 0654 Murashige and Skoog basal salt macronutrient solution (Krackeler 45-M0654-1L-EA) ~$26
[2]: Sigma M 0529 Murashige and Skoog basal salt micronutrient solution (Krackeler 45-M0529-1L-EA) ~$26
1X MS
1X MS kdorfman Wed, 12/22/2021 - 20:37*Add the other salt mixtures to the water to prevent precipitation
**pH to 5.7 with 1M KOH (initial pH = ~3.66) (needs~21 drops or ~720 µL/L)
Autoclave with stir bar in flask or bottle
Stir until cool enough to handle
Pour 30 mL per plate (use the deep ones)
[1]: Sigma M 0654 Murashige and Skoog basal salt macronutrient solution (Krackeler 45-M0654-1L-EA) ~$26
[2]: Sigma M 0529 Murashige and Skoog basal salt micronutrient solution (Krackeler 45-M0529-1L-EA) ~$26
Slime mold media
Slime mold media kdorfman Fri, 04/14/2023 - 18:18Saburaud dextrose agar
Saburaud dextrose agar kdorfman Fri, 04/14/2023 - 18:19Before you start:
- check for 20% glucose, filter sterilized; make more if needed
- warm the glucose (a ~60C oven is best; use a 37 incubator if necessary)
- sterilize a graduated cylinder if volume of glucose to be added is more than 50 mL
per L:
- 40 g glucose
- 10 g peptone
- 20 g agar
- pH 5.6
Symbiodinium
Symbiodinium kdorfman Sat, 06/22/2024 - 18:07Algae for Nematostella anemones in marine bio
culture instructions powerpoint
Subculture monthly 1 mL of old culture into 50 mL sterile f/2 medium in 125 mL erlenmeyer flask
VTM
VTM kdorfman Fri, 04/03/2020 - 14:42Viral Transport Medium
Equipment and materials needed
- Sterile Hood
- Waterbath at 56C to heat-inactivate FBS
- Sterile serological pipets
- Sterile 15 mL tubes
- Filter sterilization
Reagents
- HBSS 1X with calcium and magnesium, without phenol red
- FBS, heat inactivated
- Gentamicin sulfate (50 mg/mL)
- Amphotericin B (Fungizone) (250 ug/mL)
- Sheep blood agar plate (or equivalent) for quality control
Worm media
Worm media kdorfman Fri, 04/01/2016 - 16:28Freezing medium
Freezing medium kdorfman Fri, 04/01/2016 - 16:30Per 1 L
- 100 mL 10X M9 salts
- 240 mL glycerol
- 300 µL 1M MgSO4
- water to 1L
Filter sterilize
M9 for worms
M9 for worms kdorfman Fri, 04/01/2016 - 18:11Per 1 L:
- 100 mL 10X M salts
- 300 µL 1M MgSO4
- water to 1 L
Filter sterilize
NGM
NGM kdorfman Fri, 04/01/2016 - 16:31Per Liter of medium (~75 plates):
- 975 mL Water
- 3 g NaCl
- 2.5 g Peptone (Fisher BP1420-500 $78.80)
- 17 g Bactoagar
Autoclave with stir bar inside
Cool to 55C in a 55C water bath
Add per L (see recipes in stock solutions):
- 1 mL cholesterol (5 mg/mL in 95% EtOH)
- 1 mL CaCl2 (1 M, STERILE)
- 1 mL MgSO4 (1 M, STERILE)
- 25 mL K-phosphate buffer (1M, pH 6.0, STERILE1)
Swirl flask to mix
Dispense 10 mL into each 60mm dish.
Stack 10 high
Let stand for ~48 hours for condensation to evaporate
Pack in sterilized plastic boxes.
put bottles on stir plate near sterile hood until handle-able
-
3.3 mL K2HPO4 + 21.7 mL KH2PO4 ↩︎
Yeast media
Yeast media kdorfman Mon, 02/08/2016 - 15:03284 Yeast Plate Code
284 Yeast Plate Code kdorfman Wed, 08/23/2023 - 15:49MV + Ade
MV + Ade kdorfman Wed, 10/18/2017 - 16:42For Bio 284
- 0.15 g YNB
- 0.52 g ammonium sulfate
- 2.0 g agar
- 82 mL H2O
- 8.0 mL 1 mg/mL adenine
- 10.0 mL 20% glucose (final conc = 2g/100 mL)
Minimal Vitamin Medium plus Adenine
Label plates with "+ ADE"
Make sure it's a plus sign!
Autoclave an appropriately sized graduated cylinder to measure the glucose and adenine solutions.
Mark with two blue lines
*Yeast Nitrogen Base without amino acids and ammonium sulfate
MV
MV kdorfman Mon, 02/08/2016 - 15:04Minimal Vitamin Medium for Bio 284
Autoclave an appropriately sized graduated cylinder to measure the glucose solution.
*Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate
Mark with a single blue line
SC -Leu High Ade
SC -Leu High Ade kdorfman Sun, 01/19/2020 - 16:39Plates for Jeff Laney's Cell & Molecular Biology Lab
8X SC-HLT hi Ade concentrate
8X SC-HLT hi Ade concentrate kdorfman Fri, 01/22/2021 - 22:01Concentrate to make SC-HLT High Ade agar
add 125 mL heated concentrate to autoclaved 875 mL water + 20 g agar right out of the autoclave and still hot
Laney's SC high ade
Laney's SC high ade kdorfman Sun, 01/26/2020 - 16:22From Jeff Laney's recipe:
Per liter of medium, mix the following asceptically, and pre heat:
Component | Volume (mL) |
---|---|
10X SC-AHLT | 100 |
10X YNB+AS | 100 |
10X 20% glucose | 100 |
10X Adenine hemi sulfate | 100 |
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
Add to 20g agar in 667.5 mL water, autoclaved 30 minutes
Mark with one green line.
SC -Leu High Ade 4x + powder
SC -Leu High Ade 4x + powder kdorfman Sun, 01/26/2020 - 16:26SC -Leu High Ade 4x + powder
Use the 4x SC-AHLT to mix dry ingredients
(4x already made; not enough dry left to start over.)
per Liter:
Agar 20 g + 620 mL water. Autoclave
Meanwhile, make:
Component | Volume (mL) |
---|---|
4X SC-AHLT | 250 |
10X 20% glucose | 100 |
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
Add dry:
Component | g |
---|---|
YNB | 1.7 |
Adenine hemi sulfate | 0.2 |
Ammonium Sulfate | 5 |
Filter sterilize, warm up, then add to molten agar
Mark with one green line
SC -Leu High Ade 4X
SC -Leu High Ade 4X kdorfman Sun, 01/26/2020 - 16:23Using dilution factor 4:
(Because 10x won't go into solution)
But the agar is so viscous that bubbles don't reach the surface. Hard to pour.
Mark with one green line
SC-L high Ade from concentrate
SC-L high Ade from concentrate kdorfman Fri, 01/22/2021 - 20:55Mark with one green line.
SC-L high Ade mostly powder
SC-L high Ade mostly powder kdorfman Tue, 02/04/2020 - 13:41High concentration agar is difficult to work with.
- Tends to boil over, so needs a lot of water in the autoclave pan
- Hard to get into solution - frequently needs re-autoclaving
- So viscous that bubbles don't rise to the surface
So
- mix the ingredients that can't be autoclaved in as little water as possible
- Only use high concentration stock solutions for micro-ingredients
Per Liter:
dry ingredient | g |
---|---|
SC-AHLT | 1.64 |
YNB | 1.7 |
Ammonium sulfate | 5 |
glucose | 20 |
stock solutions | mL |
---|---|
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
10X Adenine hemi sulfate | 100 |
plus water to 150 mL
filter sterilize
Autoclave
- 20 g agar
- 850 mL water
Mix and pour
SC -Leu-low-Ade
SC -Leu-low-Ade kdorfman Thu, 01/16/2020 - 22:07SC-Leu low-Ade plates for Jeff Laney's cell and molecular biology lab
Laney's original
Laney's original kdorfman Sun, 01/26/2020 - 17:02SC-Leu low-Ade plates
From Jeff Laney's recipe:
(BUT, can't make 10X SC-AHLT)
Per liter of medium, mix the following asceptically, and pre heat:
Component | Volume (mL) |
---|---|
10X SC-AHLT | 100 |
10X YNB+AS | 100 |
10X 20% glucose | 100 |
10X Adenine hemi sulfate | 2.5 |
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
Add to 20g agar in 667.5 mL water, autoclaved 30 minutes
Mark with one green and one blue line.
SC -Leu-low-Ade 4X + powder
SC -Leu-low-Ade 4X + powder kdorfman Sun, 01/26/2020 - 17:02SC -Leu low Ade 4x + powder
Use the 4x SC-AHLT to mix dry ingredients
(4x already made; not enough dry left to start over.)
per Liter:
Agar 20 g + 617.5 mL water. Autoclave
Meanwhile, make:
Component | Volume (mL) |
---|---|
4X SC-AHLT | 250 |
10X Adenine hemi sulfate | 2.5 |
10X 20% glucose | 100 |
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
Add dry:
Component | g |
---|---|
YNB | 1.7 |
Ammonium Sulfate | 5 |
Filter sterilize, warm up, then add to molten agar
Mark with one green and one blue line
SC -Leu-low-Ade 4X
SC -Leu-low-Ade 4X kdorfman Sun, 01/26/2020 - 17:01Using 4X SC-AHLT because 10x won't go into solution:
(BUT: Agar is too viscous - bubbles won't rise to the surface before it sets up.)
Mark with one green and one blue line.
SC-AHLT low Ade mostly powder
SC-AHLT low Ade mostly powder kdorfman Tue, 02/04/2020 - 13:26High concentration agar is difficult to work with.
- Tends to boil over, so needs a lot of water in the autoclave pan
- Hard to get into solution - frequently needs re-autoclaving
- So viscous that bubbles don't rise to the surface
So
- mix the ingredients that can't be autoclaved in as little water as possible
- Only use high concentration stock solutions for micro-ingredients
Per Liter:
dry ingredient | g |
---|---|
SC-AHLT | 1.64 |
YNB | 1.7 |
Ammonium sulfate | 5 |
glucose | 20 |
stock solutions | mL |
---|---|
10X Adenine hemi sulfate | 2.5 |
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
plus water to 150 mL
filter sterilize
Autoclave
- 20 g agar
- 850 mL water
Mix and pour
Mark with one green and one blue line.
SC-AHLT
SC-AHLT kdorfman Wed, 01/15/2020 - 21:45SC minus (adenine, histidine, leucine, tryptophan)
4X will dissolve. Almost completely clear after overnight stirring. Crystal clear after autoclaving.
From the manufacturer:
Commonly added to YNB, nitrogen and glucose, at suggested g/L, for a complete yeast media. Mix with stirring for 10-15 minutes, and filter sterilize or autoclave at 121°C for 15 minutes. To prepare plates, autoclave agar separately and add to sterile medium, or add agar to liquid medium and adjust to pH 5.8-6.0 before autoclaving.
Suggested g/L: 1.64
Storage Temperature: 2-8 C
Jeff's recipe: 10X SC –AHLT 16.4 g/L
Dissolve 16.4 g of Sunrise Science SC –Adenine, –Histidine, –Leucine and –Tryptophan in 1 L distilled water and filter sterilize.
Store in the dark at 4 ˚C.
Will not go into solution.
SC-HLT Ade-free Concentrate
SC-HLT Ade-free Concentrate kdorfman Wed, 02/02/2022 - 19:18Concentrate to make SC-L High Ade or Low Ade agar
Make this concentrate, then add adenine hemi-sulfate.
for low-Ade, add 5 mg Ade per L of concentrate
for high-Ade, add 1.6 g Ade per L of concentrate
to make high-Ade from low-Ade concentrate, add 1.595g Ade per L, or 0.1595g per 100 mL
Then filter sterilize. For every L of medium, combine 20g agar autoclaved in 875 mL water with 125 mL warmed up concentrate.
add 125 mL concentrate to autoclaved 875 mL water + 20 g agar
SC-complete-high-Ade
SC-complete-high-Ade kdorfman Wed, 01/15/2020 - 22:12Liquid Medium
Using 4X SC-AHLT (because 10x won't go into solution):
Bring to final volume with water, then filter sterilize.
Jeff Laney's original (discontinued - because you can't make 10X SC-AHLT) recipe:
Component | Volume (mL) |
---|---|
10X SC–AHLT | 25 |
10X YNB+AS | 25 |
10X SC–AHLT | 25 |
20% Glucose | 25 |
10x adenine hemi sulfate | 25 |
50X Tryptophan | 5 |
100X Histidine | 2.5 |
10X Leucine | 25 |
distilled water | to final volume of 250 |
Filter sterilize
Store at room temperature
SC -Leu High Ade
SC -Leu High Ade kdorfman Sun, 01/19/2020 - 13:33Recipe from Jeff Laney. (But 10X SC-AHLT wouldn't go into solution. See 4X recipe.)
Component | Volume (mL) |
---|---|
10X SC-AHLT | 100 |
10X YNB+AS | 100 |
10X 20% glucose | 100 |
10X Adenine hemi sulfate | 100 |
50X Tryptophan | 20 |
100X Histidine HCl | 10 |
Using a different dilution factor:
Mark with one green line
YEAD
YEAD kdorfman Fri, 08/04/2023 - 22:03Yeast Extract Adenine Dextrose Medium
Ade mutants grow well on this without turning red. Better for keeping the parental strains white longer.
1 gram Yeast Extract 2 grams anhydrous dextrose (glucose) 2 grams Agar 8 mL adenine stock solution (1 mg/mL)
Adenine Stock Solution
400 mg adenine in 400 ml water (1 mg/ml) Store at room temperature. Use 2 ml stock solution for 100 ml of medium; reduce the water added to the medium by 2 ml.
Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.
YED
YED kdorfman Mon, 02/08/2016 - 19:22Yeast Extract Dextrose
For Bio 284
Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.
NOTE: 900 mL may boil over in a 1 L bottle in the autoclave. Try:
- Autoclave:
- YE + agar in 800 mL
- 100 mL H2O
- mix in BSC
- Add 100 mL sterile 20% glucose
- mix and pour
Mark with a single red line.
YEKAC
YEKAC kdorfman Mon, 02/08/2016 - 19:29Sporulation Medium for Bio 284
If you have concentrated potassium acetate solution, then:
- 20.4 mL 5M KOAc per liter
- 12.75 mL 8M KOAc per liter Fisher AAJ63372AE
Mark with a single black line.
If you are using potassium acetate powder, then:
YEPAD
YEPAD kdorfman Mon, 08/07/2023 - 17:05YEPAD medium (Yeast Extract + Peptone + Adenine + Dextrose) contains the same ingredients as YEAD but has peptone added. This is a very rich medium used for storing yeast strains.
1 gram Yeast Extract 2 grams Peptone 2 grams anhydrous dextrose (glucose) 2 grams Agar (agar-agar; gum agar) 8 mL adenine stock solution 92 ml water
YEPAD for freezing
YEPAD for freezing kdorfman Wed, 08/16/2023 - 18:06Yeast strains, transformed or untransformed, can be maintained as colonies on solid media at 4° and restreaking every 2 to 4 weeks. Alternatively, strains may be stored at -80 indefinitely. This is preferable in that it reduces the likelihood of accumulating spontaneous mutations.
To make frozen stocks of yeast strains: -Use sterile technique and sterile solutions throughout this method.-
Grow a starter culture at 30 with shaking (250 rpm) until it reaches saturation.
In a 1.8 ml cryotube, mix 0.5 ml of the saturated culture with 0.5 ml of YPD containing 20% glycerol.
Flash freeze the tube in liquid nitrogen and store at -80.
To use, chip out a few pieces of the frozen stock using a sterile pipette tip or sterile toothpick and streak onto a plate containing the appropriate solid media.
Allow the yeast to grow at 30 until colonies appear (2-6 days).
YNB + AS
YNB + AS kdorfman Wed, 01/15/2020 - 21:4410X YNB+AS
17 g/L YNB and 50 g/L AS
Dissolve 17 g Difco Yeast Nitrogen Base w/o Amino Acids and w/o Ammonium Sulfate and 50 g Ammonium Sulfate in 1L distilled water.
Autoclave for 15 min.
Store at room temp.
YPD
YPD kdorfman Mon, 02/08/2016 - 15:04Yeast extract-Peptone-Dextrose medium:
Dilutions
Dilutions kdorfman Mon, 03/07/2016 - 21:07Enter initial and final concentration (in matching units!), and final volume
Fixatives
Fixatives kdorfman Tue, 06/16/2009 - 17:53How to fix cells and tissues
Cnidocyte fixation solution
Cnidocyte fixation solution kdorfman Thu, 10/10/2024 - 17:53Cnidocyte fixation solution
Combine 872 µL of 12 PSU ASW,
108 µL of 37% (vol/vol) formaldehyde
20 µL of 0.5 M EDTA.
Store the solution at 4 °C for up to 6 months.
Fixing adherent cells
Fixing adherent cells kdorfman Thu, 05/21/2020 - 17:39To fix the cells grown on a coverslip
- Work in a fume hood
- Gently rinse cells 2X in warm PBS (remove medium, add PBS, repeat)
- Leave cells in this PBS—until you add the fixative.
- Remove final PBS rinse, add about 1 mL of fixative to the cells
- Fix cells in hood for 10 mins.
- Rinse cells by dunking each coverslip in 3 beakers of PBS-Tween-Azide, 10 X per beaker.
- Place coverslip back in dish with fresh PBS-Tween-Azide
- If you are not staining immediately, store cells in PBS-Tween-Azide in fridge.
Remember:
- Work in the fume hood
- Wear gloves!
- Keep track of which side of the coverslip the cells are on
- Discard used fixative in the appropriate hazardous waste container in the hood.
Formaldehyde fix
Formaldehyde fix kdorfman Thu, 07/05/2012 - 20:55Make in fume hood
3.7% formaldehyde
0.5% Triton X
in PBS
Formaldehyde stock: 37%
Triton X stock: 10%
Methanol fix
Methanol fix kdorfman Tue, 07/24/2012 - 17:05100% methanol at ice temperature
10 min
No permeabilization step needed.
Wash with PBS afterwards.
The methanol fixation is an easy method; however, it frequently solubilizes and removes membrane bound antigens. By a simple precipitation of the protein, methanol only provides low structural preservation.
PFA 4% in 1/3 SW
PFA 4% in 1/3 SW kdorfman Thu, 10/10/2024 - 15:394% paraformaldehyde in 1/3 ASW
Paraformaldehyde stock (EMS RT 15714): 32%
PFA 5% for fish
PFA 5% for fish kdorfman Wed, 10/02/2019 - 16:565% paraformaldehyde
in PBS
Paraformaldehyde stock: 32%
Paraformaldehyde Fix
Paraformaldehyde Fix kdorfman Sun, 10/27/2013 - 19:323.7% paraformaldehyde, 0.5% Triton-X
in PBS
To fix the adherent cells (still in hood):
Paraformaldehyde stock: 32%
Triton X stock: 10%
EMS RT 15714
Paraformaldehyde from powder
Paraformaldehyde from powder kdorfman Wed, 03/03/2021 - 20:12Stir, heat to ~60C
Add NaOH until it clears, then bring to final volume
Paraglut
Paraglut kdorfman Mon, 09/24/2012 - 16:123.2% paraformaldehyde
0.1% glutaraldehyde
0.5% Triton X-100
in PBS
Bring to final volume with distilled water.
Gels
Gels kdorfman Thu, 05/24/2012 - 17:17EtBr gel
EtBr gel kdorfman Tue, 09/20/2022 - 15:02Ethidium bromide gel
*Add weighed agarose to measured TAE in a flask (about half the maximum volume of the flask)
*Boil in the microwave CAREFULLY (power level 0.5) until completely dissolved (check by swirling) DO NOT LET IT BOIL OVER
*Allow to cool slightly
*Add EtBr (in the fume hood)
*Aliquot into 50 mL conical tubes
*Put in rack in 65C waterbath
Clean-up
Rinse gel rigs within an inch of their lives
Put gels into zip lock bag, stick a hazardous waste sticker on it, schedule hazardous waste pickup (sign into CEMS)
pour used buffer into gallon jug with EtBr removing "tea bag", stir overnight, then pour down the drain
Gel Rigs
Gel Rigs kdorfman Wed, 01/11/2023 - 20:25brand | room | L x W (cm) | gel vol | # small wells | small well uL | # large wells | large well uL |
---|---|---|---|---|---|---|---|
IBI | 264 | 10 x 7 cm | 50 mL | 15 | 25 | 8 | 50 |
BioRad | 360, 364, 368 | 10 x 7 cm | 50 mL | 15 | 25 | 8 | 50 |
Labnet | genetics | 10 x 15 cm | 100 mL | 20 | 30 | 16 | 50 |
Sybr Safe gel
Sybr Safe gel kdorfman Tue, 09/20/2022 - 15:011 uL /10 mL gel
*Add weighed agarose to measured TAE in a flask (about half the maximum volume of the flask)
*Boil in the microwave CAREFULLY (power level 0.5) until completely dissolved (check by swirling) DO NOT LET IT BOIL OVER
*Allow to cool slightly
*Add SYBR-safe
*Aliquot into 50 mL conical tubes
*Put in rack in 65C waterbath
Clean-up
Everything can go in the trash or down the drain
Hormones & Inhibitors, etc.
Hormones & Inhibitors, etc. kdorfman Wed, 06/20/2018 - 21:25By compound
By compound kdorfman Mon, 04/23/2018 - 17:49ATP
ATP kdorfman Mon, 04/23/2018 - 17:5010 mM ATP Stock
(from Maniotis)
- 60 mg ATP in 8 mL H2O
- pH to 7.0 with 0.1 M NaOH
- Bring volume to 10 mL with H2O
- Aliquot and freeze.
Angiotensin
Angiotensin kdorfman Thu, 04/26/2018 - 17:10Sigma A9525 1mg/mL in water
Vasoconstrictor
Soluble in water 25 mg/mL (only use sterile water!)
MW: 1046.18 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
Subject to degradation in freezer storage - wear gloves!
Make 1 mg/mL stock (= 1.0462 mM)
Working concentration 1 µM
Dissolve 1:1000 in HBS
BMS
BMS kdorfman Fri, 03/25/2022 - 13:50BMS 833923
___ | ___ |
---|---|
CAS Registry No. | 1059734-66-5 |
Formal Name: | N-[2-methyl-5-[(methylamino)methyl]phenyl]-4-[(4-phenyl-2-quinazolinyl)amino]-benzamide |
Synonym | XL 139 |
MF: | C30H27N5O |
FW: | 473.6 |
Purity: | ≥98%S |
Stability: | ≥2 years at -20°C |
Solubility | 0.1 mg/ml in a 1:7 solution of ethanol:PBS (dissolve in ethanol first!) Use aqueous solution within 1 day |
Supplied as: | A crystalline solid |
UV/Vis. | λmax: 315 nm |
Smoothened (Smo) is a cell surface receptor that, with Patched, mediates sonic hedgehog (Shh) signaling to regulate gene expression through the Gli transcription factors.1 BMS 833923 is an orally bioavailable inhibitor of Smo.2,3 It blocks binding of BODIPY cyclopamine (IC50 = 21 nM) and inhibits Gli activation in cell lines that express wild-type Smo or activated mutant forms of Smo (IC50s = 6-35 nM). BMS 833923 robustly inhibits Shh pathway activity and prevents tumor growth in medulloblastoma and pancreatic carcinoma xenograft models.
References
Ruiz-Gómez, A., Molnar, C., Holguín, H., et al. The cell biology of Smo signalling and its relationships with GPCRs. Biochim. Biophys. Acta1768(4), 901-912 (2007).
Sandhiya, S., Melvin, G., Kumar, S.S., et al. The dawn of hedgehog inhibitors: Vismodegib. J. Pharmacol. Pharmacother.4(1), 4-7 (2013).
Lin, T.L. and Matsui, W. Hedgehog pathway as a drug target: Smoothened inhibitors in development. OncoTargets and Therapy5, 47-58 (2012)
BP-A
BP-A kdorfman Mon, 03/22/2021 - 18:11Bisphenol A
Sigma 239658
MW 228.29
100 mM stock in DMSO = 0.023g/mL
BP-S
BP-S kdorfman Mon, 03/22/2021 - 18:12Sulfonyldiphenol
Sigma 103039
MW = 250.27
100 mM stock in DMSO = 0.025g/mL
Blebbistatin
Blebbistatin kdorfman Mon, 04/23/2018 - 17:49Myosin inhibitor
(-)-Blebbistatin Sigma B05650-1mg
Info from Cayman:
A stock solution may be made by dissolving the (±)-blebbistatin in an organic solvent purged with an inert gas. (±)-Blebbistatin is soluble in organic solvents such as DMSO and dimethyl formamide (DMF). The solubility of (±)-blebbistatin in these solvents is approximately 10 mg/mL.
(±)-Blebbistatin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, (±)-blebbistatin should first be dissolved in DMF and then diluted with the aqueous buffer of choice. (±)-Blebbistatin has a solubility of approximately 0.5 mg/ml in a 1:1 solution of DMF:PBS (pH 7.2) using this method. We do not recommend storing the aqueous solution for more than one day.
(±)-Blebbistatin is a selective cell-permeable inhibitor of non-muscle myosin II ATPases.1,2 It rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (IC50 = 0.5-5.0 μM), while poorly inhibiting smooth muscle myosin (IC50 = 80 μM).3 Through these effects, blebbistatin blocks apoptosis-related bleb formation, directed cell migration and cytokinesis in vertebrate cells. Blebbistatin is inactivated by UV light,4 which may be particularly important in fluorescent cell imaging applications.
Note green fluorescent crystals seen in a 75µM solution.
Stock was 1 mg/mL in DMSO
44 µL 1mg/mL blebbistatin in DMSO in 1 mL non-CO2 medium
REDO
1 mg in 34 µL DMSO = ~100mM
MW = 292
Use at ~150 µM
Bradykinin
Bradykinin kdorfman Mon, 04/23/2018 - 18:56- 9-amino acid peptide chain: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Bradykinin raises internal calcium levels
*Sigma B3259 - 1 mg septum bottle
MW = 1060.2
1 mg/mL stock
(= 1.0602 mM)
Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)
Aliquots are 21.2 µL.
2 µM solution for use
(=2.12 µg/mL)
Brefeldin A
Brefeldin A kdorfman Tue, 11/12/2019 - 20:48Fisher 00-4506 1000x solution
Keep in refrigerator
Description
Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of Brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines. Brefeldin A is effective for enhanced detection of a majority of mouse and human intracellular cytokines; however, it is advised that the investigators evaluate the use and efficacy of this reagent as well as other protein transport inhibitors such as Monensin in their specific assay system.
Applications Reported
The Brefeldin A Solution is supplied at 1000X working concentration in methanol. Prior to addition to the cell cultures, a 1:1000 dilution in the culture media should be made. The final working concentration of the Brefeldin A (at 1x) is 3.0 ug/ml.
Caffeine
Caffeine kdorfman Tue, 04/13/2021 - 15:25MW 194.19
Soluble 10 mg/mL in water (with heat and stirring)
Make a 50mM (=0.05M) solution: 9.71mg/mL
Cannabinol
Cannabinol kdorfman Fri, 05/11/2018 - 16:11Tetrahydrocannabinol solution 1 mg/mL in methanol
Sigma T464
store at 4C
Vial is in a white box in fridge door, room 362A
Centrinone
Centrinone kdorfman Thu, 06/01/2023 - 18:38PLK4 (Polo-like kinase) inhibitor
Plk4 has been identified as a master regulator of centriole replication, and its aberrant expression is closely associated with cancer development
PLK4 functions downstream of ROCK2 to drive centrosome amplification in arrested cells. PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.
100uM stock
use 125 nM for HeLa (2.5 uL stock in 2 mL medium)
use 300 nM for LLCPk-1 (6 uL stock in 2 mL medium)
Concanavalin A
Concanavalin A kdorfman Mon, 04/23/2018 - 18:21- Sigma C5275 5 mg
- Plant mitogen
- Lectin (carbohydrate-binding protein) extracted from the jack-bean, Canavalia ensiformis
Con A binds specifically to α-Mannose, α-Galactose structures found in sugars, glycoproteins and glycolipids
Make stock 5 mg/mL solution (add 1 mL sterile PBS to bottle – gently rotate to mix)
Keep sterile - work in sterile hood
aliquot 100 µL into microfuge tubes (cannot take repeated freeze-thaw cycles)
Con A coverslips
Con A coverslips kdorfman Sun, 05/24/2020 - 15:38Requirements: * Acid washed coverslips stored in 100% ethanol.
ConA 0.5 mg/mL.
Large petri dishes to store the coverslips.
Procedure:
Flame the acid-washed coverslips and cool for 10 sec.
(Make 9-14 coverslips at a time to save time.)Place the coverslips on top of the large petri dish.
Thaw the conA from the freezer and dilute to 0.5 mg/mL
apply around 400uL to the first two coverslips
Wait for 10 sec.
Tilt the coverslips and suck out the excess ConA
Repeat the same for all the coverslips.
Cover the petri dish with another lid of petri dish and allow a small opening to dry.
If possible,use a vacuum pump to dry it faster.
The process usually takes 15 minutes and drying takes half an hour.
Storage:
* In a petri dish, place the coverslips on top of a clean kimwipe with the conA surface facing up.
Cover with the lid. 10 min before needed, UV sterilize them.
Alternatively, UV sterilize all of them after drying and open the dishes only in aseptic conditions or inside hood.
Con A dishes
Con A dishes kdorfman Sun, 05/24/2020 - 15:37Concanavalin-coated coverslip dishes
Start with 5 mg/mL ConA stock solution
Keep sterile - work in sterile hood
aliquot 100 µL into microfuge tubes (cannot take repeated freeze-thaw cycles)
Coat coverslip bottom dishes with either:
- 0.75 mL 0.1 mg/mL: 0.015 mL (5 mg/mL) + 0.735 mL PBS
- 0.75 mL 0.5 mg/mL: 0.075 mL (5 mg/mL) + 0.675 mL PBS
Incubate the coverslip dishes for 1 hour 37˚C
Remove the solution
rinse in PBS
Air dry the dish.
Cyclin Inhibitor
Cyclin Inhibitor kdorfman Wed, 04/25/2018 - 19:55CKD1 inhibitor IV RO3306
1 mM in DMSO
A cyclin-dependent kinase inhibitor protein inhibits cyclin-dependent kinase. Several function as tumor suppressor proteins. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors (called CDIs, CKIs or CDKIs). CDIs are involved in cell cycle arrest at the G1 phase.
Cycloheximide
Cycloheximide bcrcstaff Thu, 03/21/2019 - 13:28Protein synthesis inhibitor
Fisher AC357420010
MW = 281.35 g/mol
C15H23NO4
Solubility in water: 2.1 g/100mL (
soluble in chloroform, ether, acetone, methanol, and ethanol
powder
Cyclopamine
Cyclopamine kdorfman Fri, 03/25/2022 - 13:4310 mM
Store in freezer
C27H41NO2
MW = 411.62
Soluble 6.86 mg/mL in DMSO (warm to 37C and shake to get it into solution)
Reaction Conditions :
- 20 μM, 48 hours for cell yield inhibition
- 10 μM, 48 hours for apoptosis induction (measured by PARP expression)
Cyclopamine is a naturally occurring Hedgehog (Hh)?specific small?molecule signaling steroidal alkaloid inhibitor, causes a profound inhibition of tumor growth, has significant anti?invasive, anti?proliferative and anti?estrogenic potency in human breast cancer cells [2] [1]. The EC50 of cyclopamine is 10.57 μM, it was identified by an FXR-bla (farnesoid X receptor- b-lactamase) assay [3].
Hh signaling pathway plays a critical role in embryonic development and tumorigenesis [4]. Hh signaling pathway shows saliency in regulating cellular proliferation and differentiation in a wide array of human tissues. It is related to aberrant cell survival in numerous human malignancies, ranging from BCCs and medulloblastomas to small cell lung, gastrointestinal, breast and prostate tumors [1].
Treated with cyclopamine (10 or 20 μM) only and incubated for time periods ranging from 0 to10 days, MCF-7 cells and MDA?MB?231 cells displayed a significant reduction in proliferation rate compared with the control cells on days 3 and 6 (P
Embryoes exposed to cyclopamine resulted in visible external defects, including cyclopia, proboscis formation, microphthalmia, thoracic lordosis, amelia and decreased body size. Examination of gastrointestinal organs revealed severe deficits, including less length of the gut tube and mesenchymal cell numbers in foregut-derived organs. Ectopic structures in duodenum, stomach, and dorsal pancreas were also found [5].
Cytochalasin D
Cytochalasin D kdorfman Mon, 04/23/2018 - 18:21MW = 507.62
Sigma C2618: 200 µL 5 mg/mL (~10 mM) in DMSO
Sigma C8273: 5 mg. Dissolve in 1 mL DMSO
- alkaloid mycotoxin produced by Helminthosporium and other molds.
- disrupts actin microfilaments
- binds to F-actin polymer
- prevents polymerization of actin monomers
- Aliquot 10 µL 10 mM
- Add 90 uL DMSO (makes 1 mM)
- Dilute this 1:1000 into medium (makes 1uM)
Dilute the 1 uM to desired working concentration
Store in the dark, in the freezer
Adding 490 µL of non-CO2 medium makes 500 µL of 200 µM
Final dilution should contain no more than 0.1% DMSO
250 nM in the dish should give an effect
15 - 30 min
DAPT
DAPT kdorfman Fri, 03/25/2022 - 18:16Biological Activity for DAPT (Info from Tocris 2634)
Purchased from Fisher: AAJ65864MA
Mfr: Thermo Scientific Chemicals J65864MA
M. Wt: 432.46 Formula: C23H26F2N2O4 Store at +4°C Light sensitive
Solubility: 43.24 mg/mL (100 mM) in DMSO
DAPT is a γ-secretase inhibitor. DAPT reduces Aβ40 and Aβ42 levels in human primary neuronal cultures (IC50 values are 115 and 200 nM for total Aβ and Aβ42 respectively) and in brain extract, cerebrospinal fluid and plasma in vivo. DAPT has no effect on APPα and APPβ levels. DAPT blocks Notch signaling in hybrid human-mouse fetal thymus organ culture (FTOC) and causes ESCs to commit to neuronal differentiation. DAPT can be used in a small molecule cocktail to derive cortical neurons from hPSCs and to maintain hepatocytes in culture. DAPT also promotes the formation of cone photoreceptors in retinal organoids.
Estradiol
Estradiol kdorfman Thu, 03/24/2022 - 17:40β17-Estradiol
(Purchased 2022 for NAP lab Bio 388)
500 mg
Store powder at -20
Make 0.05M stock solution: 0.068 g in 5 mL DMSO. vortex
__ | __ |
---|---|
CAS RegistryNo | 50-28-2 |
Formal Name | estra-1,3,5(10)-triene-3,17β-diol |
Synonyms | β-Estradiol, Estradiol, 17β-Oestradiol, E2 |
MF | C18H24O2 |
FW | 272.4 |
Purity | ≥98% |
Stability | ≥2 years at -20°C |
Supplied as | A crystalline solid |
UV/Vis | λmax: 281 nm |
solubility | 2.5 mg/mL in ethanol 20 mg/mL in DMSO 0.2 mg/mL in a 1:4 solution of DMSO:PBS (pH 7). Dissolve in DMSO first! Use within a day |
Estrogens direct the development of the female genotype in embryogenesis and at puberty. 17β-Estradiol is the major estrogen secreted by the premenopausal ovary. It is synthesized from testosterone primarily in the ovarian granulosa cells and placenta, but small amounts can be produced in the adrenal gland.1,2 Plasma 17β-estradiol levels increase gradually between days 1-7 of the menstrual cycle followed by a sharp increase to a peak value of about 300 pg/ml on day 12, just prior to ovulation
Fibronectin
Fibronectin kdorfman Thu, 04/26/2018 - 17:15Sigma F1141
Attachment factor
GSA 10
GSA 10 kdorfman Thu, 01/28/2021 - 18:39Smo (Smoothened) receptor* agonist
Tocris 4918 10 mg
MW = 459.94
Make a 5 mM stock solution in 4.34 mL DMSO (heat to 50C to dissolve)
*Non-classical G-protein-coupled receptors that belong to the Frizzled family. Smoothened receptors lack the ability to directly interact with their endogenous ligand, Hedgehog (Hh). In the resting state, Smo receptors are bound to patched (Ptc).
Does not recognize the classic cyclopamine (Cat. No. 1623) binding site. Does not promote Smo translocation to the primary cilium; is strongly potentiated by forskolin and cholera toxin. Displays anti-adipogenic effects in vitro, mediated by a non-canonical Hedgehog signaling pathway. Promotes differentiation of multipotent mesenchymal progenitor cells into osteoblasts.
H-89
H-89 kdorfman Mon, 03/22/2021 - 18:08H 89 hydrochloride Tocris 2910
- Protein kinase A inhibitor
- Also inhibits several other kinases (IC50 values are 80, 120, 135, 270, 2600 and 2800 nM for S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b).
- Exhibits antinociceptive activity.
- Enhances survival and clonogenicity of dissociated human ESCs through ROCK inhibition.
Store in freezer
1 mg
MW 532.79
1 mg/mL in DMSO
HU (Hydroxyurea)
HU (Hydroxyurea) kdorfman Wed, 01/06/2021 - 18:29ingredient | working conc | MW | stock |
---|---|---|---|
HU | 0.2 M | 76 | Koshand lab 2M in H2O |
Hydroxyurea: (Sigma H8267-1g)
- To make 50 mL YPD with 0.2M HU
vi = (0.2M * 50 mL)/2M = 5 mL
100µL 2M stock per mL medium
Buy 1 g, make 6.6 mL 2M stock
- MW = 76, so 1 g = 1/76 mol = 0.0132 mol
- vol = (1000 mL/2 mol) * 0.0132 mol = 6.6 mL
See yeast recipes for QBoC https://wahoo.cns.umass.edu/node/554/
Haloperidol
Haloperidol kdorfman Thu, 03/24/2022 - 20:06TCI H0912
C21H23CIFNO2
MW 375.87
Solubility:1
- 0.1 M HCl: 3 mg/mL
- DMSO: soluble
- H2O: insoluble
- ethanol: soluble (only in theory - in practice, not so much)
Make 10 mL 10mM stock solution: 0.376g in 10 mL DMSO. Vortex to mix
Haloperidol has been used:
- in ethanol to serves as an inhibitor of Erg2p
- to address the mechanism of haloperidol in ferroptosis using hepatocellular carcinoma cells: Hep G2 and Huh-7 cell lines
- in receptor internalization assay
- as an antipsychotic drug in Dulbecco′s Modified Eagle medium
Biochem/physiol Actions:
Haloperidol is a butyrophenone antipsychotic. It is also classified as a neuroleptic (powerful tranquilizer). Haloperidol acts as a D2, D3, and D4 dopamine receptor antagonist and thus causes Parkinson′s disorder. It also has a negative effect on the central nervous system.
Hydrocorisone
Hydrocorisone kdorfman Mon, 03/22/2021 - 18:06Fisher A16292 (Alfa Aesar) 1 g
1 mg/mL in 100% ethanol.
IGF-1R I
IGF-1R I kdorfman Fri, 03/25/2022 - 14:37Insulin-like growth factor-1 receptor inhibitor
Molecular Formula: C25H26ClN5O3
Formula Weight: 479.96
Solublity: 96 mg/mL in DMSO
IWP-2
IWP-2 kdorfman Fri, 03/25/2022 - 16:47Selleck Chemical S7085 (Fisher NC0736836)
IWP-2 is an inhibitor of Wnt processing and secretion with IC50 of 27 nM in a cell-free assay, selective blockage of Porcn-mediated Wnt palmitoylation, does not affect Wnt/β-catenin in general and displays no effect against Wnt-stimulated cellular responses. IWP-2 specifically inhibits CK1δ.
Selective inhibitor of Porcn-mediated Wnt secretion.
IWP-2 is useful in both regenerative medicine and anticancer efforts. IWP-2 inactivates Porcn, a membrane-bound O-acyltransferase (MBOAT), and selectively inhibits palmitoylation of Wnt. IWP-2 blocks Wnt-dependent phosphorylation of Lrp6 receptor and Dvl2, and β-catenin accumulation.
Soluble in DMF at 12.5 mg/mL (26.79 mM) Insoluble in water and DMSO
Concentration | 1 mg/ | 5 mg/ | 10 mg/ |
---|---|---|---|
1 mM | 2.1432 mL | 10.7158 mL | 21.4316 mL |
5 mM | 0.4286 mL | 2.1432 mL | 4.2863 mL |
10 mM | 0.2143 mL | 1.716 mL | 2.1432 mL |
50 mM | - | - | - |
Importazole
Importazole kdorfman Thu, 05/10/2018 - 14:25401105-10mg Calbiochem
Importazole is an inhibitor of importin-β transport receptors.
During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. Importazole somehow disrupts the importin/RAN interaction. Importazole is selective among other transporters. Compounds are imported into, but not out of, cells.
A cell-permeable diaminoquinazoline compound that selectively blocks importin-β-mediated nuclear import of NLS bearing cargos in a reversible manner.
Soluble in DMSO (25 mg/ml; clear, colorless solution)
5 mg/mL ailquots in freezer
Ionomycin
Ionomycin kdorfman Mon, 04/23/2018 - 18:47Raises intracellular calcium levels
Fisher or Invitrogen (Life Technologies) I24222
1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.
3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL
24 µL in 8 mL, aliquot 800 µL
They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.
3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
45 µL in 15 mL
Takes a long time to thaw. Vortex.
Aliquot 1.55 mL so they can do two 750 µL experiments
Jasplakinolide
Jasplakinolide kdorfman Thu, 04/26/2018 - 17:44(out of stock 2018)
- Jasplakinolide – Santa Cruz biochemical, product sc202191 F
- inducer of actin polymerization & stabilization; inhibits actin filament disassembly
- F-actin probe
- MW = ~710
- powder is stable frozen over a year
- stock is stable 3-4 mo at -20C
- Make 1 mM stock with 50 µg in 70 µL DMSO
- Add 5 mL non-CO2 medium to make ~14 µM
- 50 nM to 5 μM working range
- minutes to hours incubation time
LDL
LDL kdorfman Fri, 05/11/2018 - 16:29Low-density lipoprotein from human plasma, Dil complex (Dil LDL)
1 mg/mL
200 uL
DO NOT FREEZE
Life Technologies L3482
Laminin
Laminin kdorfman Thu, 04/26/2018 - 18:16Laminin from mouse BD354232
1 mL 1 mM
Cell adhesion
Latrunculin
Latrunculin kdorfman Thu, 04/26/2018 - 17:46Alexis Biochemicals 350-036-C100 Catalog # t110
Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes
Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.
Inactivated by FBS. (Rinse medium off before treatment.)
MW = 395.5
Soluble in DMSO and ethanol.
Store in freezer.
Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM
(2.5 µM = 25 x 100 nm)
Stock is 1mm
µL 1mM stock | µL serum-free buffer or medium | µM final concentration |
---|---|---|
1 * | 99 | 10 |
1 | 399 | 2.5 |
1 | 999 | 1 |
*Use this to make further dilutions
ML-7
ML-7 kdorfman Wed, 04/25/2018 - 19:42ML-7 Sigma, 12764.
selective myosin light chain kinase inhibitor
- soluble 10mg/mL in 50% EtOH
- MW = 452.74
- 5 mg in bottle
- add 1100 µL to make 10 mM solution
- used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
- 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
- make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM
Melanocyte Stimulating Hormone
Melanocyte Stimulating Hormone kdorfman Wed, 04/25/2018 - 20:48Sigma M4135 1 mg
Molecular Weight 1664.88
From Sigma: Amino Acid Sequence:
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
Pituitary hormone which causes darkening skin pigmentation from amphibians to humans. In mammals, it can also have behavioral effects on learning, attention, and memory.
Hormone that stimulates melanogenesis; facilitates learning and memory; affects inflammatory and immune responses and peripheral nerve regeneration.
α-Melanocyte-stimulating hormone (α-MSH) acts as an anti-inflammatory agent via down regulating the production and activity of the pro-inflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF)-α and IL-6 expressed in various cells of the immune system. It also controls the nitric oxide production associated with inflammation. α−MSH inhibits nuclear factor-κB (NF-κB)-dependent gene transcription and NF-κB pathway induced by TNF and other inflammatory agents. This activity of α-MSH is mediated through the production of cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) enzyme. α–MSH functions as a potent therapeutics for various conditions resulted through NF-κB activation including, inflammatory diseases, human immunodeficiency virus (HIV) replication in AIDS (acquired immunodeficiency syndrome), and septic shock.[2] α-MSH has an essential role to play in melanin production in animals. α-MSH regulates development of several skin diseases, including cutaneous inflammation and hyper-proliferative skin diseases.[3] Linkage
Derived from ACTH 1-13 Other Notes
Lyophilized from 0.1% TFA in H2O Packaging
1, 5 mg in glass bottle Application
α-Melanocyte stimulating hormone (α–MSH) has been used for following studies: • it has been Intracerebroventricularly (icv) injected to mice for behavioral studies.[1] • to determine the effect of α−MSH on growth of stationary Nb 2 node lymphoma cell cultures.[4] • to determine the effect of α−MSH on leptin secretion in the primary cultures of differentiated adipocytes.[5] • α−MSH promoted melanin production in the B16-F1 cells from murine melanoma cell line.[6] General description
α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide, mostly produced by the cells in the brain, pituitary and circulation.[2] Pro-inflammatory cytokines or UV light induced epidermal cells such as keratinocytes and melanocytes synthesize and discharge α–MSH. Poopiomelanocortin (POMC) acts as a precursor for α-Melanocyte-stimulating hormone (α-MSH) production.[3]
Monastrol
Monastrol kdorfman Thu, 05/10/2018 - 15:17Inhibits kinesin 5
200 mM in DMSO
Final concentration 200 uM
5.5 uL aliquots
Monensin
Monensin kdorfman Wed, 04/25/2018 - 21:25Monensin sodium salt
Sigma M5273
soluble in methanol 50 mg/mL
insoluble in water
soluble in DMSO, ethanol
ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells
inhibits transition from G1 to S
antiprotozoal, antibacterial, or antifungal agent
MW 692.9
1 µM = half-maximal inhibitory effect
Nerve Growth Factor
Nerve Growth Factor kdorfman Fri, 05/11/2018 - 16:16Nerve Growth Factor 2.5S
Harlan.com Cat no. 5025
From Sigma: 2.5S subunit of nerve growth factor-7S (NGF-7S) is essentially the β-subunit when isolated from male mouse submaxillary glands under initially dissociative conditions by a modification of the method of Bocchini and Angeletti.
Stored at 4C
Nocodazole
Nocodazole kdorfman Mon, 04/23/2018 - 17:51Prevents polymerization of microtubules. Prevents cells from entering metaphase.
Acros 358240100, 10 mg
Soluble in DMSO
Dilute stock in medium to treat cells.
Make a 10 mg/mL (=33mM) stock: Add 1 mL DMSO to 10 mg in bottle.
Aliquot 3 µL 33 mM per tube.
Working concentration range is usually 100nM - 30 µM.
add 97 µL DMSO to tube to make 100 µL of 1 mM
33 µL 1mM + 10 mL medium = 3.3 µM
Oxidopamine
Oxidopamine kdorfman Fri, 03/25/2022 - 15:41Oxidopamine (hydrobromide)
Selleck Chemicals S5324-25mg (Fisher 17159569)
MW = 250.09
Formula: C8H11NO3.HBr
Oxidopamine (6-hydroxydopamine, 6-OHDA, 2,4,5-trihydroxyphenethylamine) is a neurotoxic synthetic organic compound that acts as an antagonist of the neurotransmitter dopamine with potential antineoplastic activity. Solutions should be freshly prepared and protected from exposure to light.
solvent | mg/mL | mM |
---|---|---|
DMSO | 50 mg/mL | (199.93 mM) |
Ethanol | 50 mg/mL | (199.93 mM) |
Water | 50 mg/mL | (199.93 mM) |
Concentration | 1 mg/ | 5 mg/ | 10 mg/ |
---|---|---|---|
1 mM | 3.9986 mL | 19.9928 mL | 39.9856 mL |
5 mM | 0.7997 mL | 3.9986 mL | 7.9971 mL |
10 mM | 0.3999 mL | 1.9993 mL | 3.9986 mL |
50 mM | 0.0800 mL | 0.3999 mL | 0.7997 mL |
PMSF
PMSF bcrcstaff Thu, 03/21/2019 - 14:14PhenylMethylSulfonyl Fluoride
Serine protease inhibitor
Sigma https://www.sigmaaldrich.com/catalog/product/sigma/p7626
Plant hormones
Plant hormones bcrcstaff Thu, 03/21/2019 - 14:49Compound | function | Form | Room | storage | stock | Cat # |
---|---|---|---|---|---|---|
ABA Plant hormone involved in growth and stress response | powder | 362A | fridge | 50 mM in EtOH | Fisher AC133480010 | |
ABA | powder soluble 20 mg/mL in DMSO, ethanol | 362A | freezer | PlantMedia 30631017-1 soluble in methanol |
||
Ascorbic acid | Enzyme cofactor | Powder | 362A | RT | Fisher S26184 | |
Caffeine | Growth inhibitor | Powder | 362A | RT | Fisher ICN15011483 | |
Chitosan medium viscosity | Component of fungal cell walls and arthropod exoskeletons | Powder | 362A | RT | Bio-World 40300161-1 | |
Cycloheximide | Protein synthesis inhibitor | Powder | 362A | RT | Fisher AC357420010 | |
Gibberellic acid | breaks seed dormancy | 13 mg/mL (=37.5mM) solution | 362A | 4C | PlantMedia 30631025-1 | |
IAA | auxin growth regulator stimulates root growth |
solid | 362A | 4C | 100 mM in EtOH | PlantMedia 30631010- |
Imidacloprid (pestanal) | Systemic insecticide | neat | 362A | dessicator | Sigma 37894 | |
kinetin | Plant cytokinin | Powder | 362A | RT | Sigma K0753 | |
Lactic acid | Plant growth stimulator | 69% soln aq | 362A | dessicator | Fisher AC250300100 | |
Methyl jasmonate | Pathogen response | Liq | 362A | RT | PlantMedia30631015-3 | |
Nicotine | Plant defense chemical | liq | 362A | dessicator | Fisher AC181420050 | |
Salicylic acid | Response to abiotic stress | Powder | 362A | RT | 100 mM in EtOH | Fisher S25515 |
Selenium | varies | Powder | 362A | RT | Strem 93-3416 |
Poly-L-Lysine
Poly-L-Lysine kdorfman Thu, 04/26/2018 - 17:04Non-specific attachment factor for cells; use it to coat coverslips.
Sigma P1524
- Add 50 ml of sterile tissue culture grade water to 5 mg of poly-lysine.
- Aseptically coat culture surface with 1 ml per 25 cm2 of solution. Rock gently to ensure even coating of the culture surface.
- After 5 minutes, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water.
- Allow to dry at least two hours before introducing cells and medium. If glassware or slides must be sterilized after coating with poly-lysine, γ-irradiation is recommended instead of autoclaving.
Can also buy poly-lysine coated slides: Fisher 6776215
Purmorphamine
Purmorphamine kdorfman Thu, 01/28/2021 - 18:25Tocris 4551 10 mg
MW = 525.12
Make a 10 mM stock in DMSO in 1.9 mL DMSO
Retinol
Retinol kdorfman Wed, 04/27/2022 - 17:09Fisher J62079
Store in freezer; hygroscopic
C20H30O
MW = 286.459
General description (from Fisher)
- All-trans-retinol, commonly known as Vitamin A, is a fat-soluble essential nutrient
- All-trans-retinol is involved in hormonal signaling via the retinoid receptors
- It is an important regulator of cell division and apoptosis
- Deficiency can lead to vision, bone, immune, and skin disorders
Practically insoluble aq. soln. or glycerol; soluble in absolute alcohol, methanol, chloroform,ether,fats and oils.
Can make a 50 mg/mL solution in 95% ethanol, but it's very hard to weigh
From Sigma information sheet:
SOLUBILITY / SOLUTION STABILITY:
- RE is practically insoluble in water or glycerol. It is soluble in absolute ethanol, methanol, chloroform, ether, fats and oils.
- RE has been dissolved at 50 mg/ml in chloroform; a clear yellow to orange solution results.
- Stock solutions of RE (1 mg/ml) were prepared in ethanol, diluted in DMSO under low light conditions and stored at -50°C under nitrogen in brown glass vials.
- RE solutions (50 μM) were sterile filtered before use.
- RE both as a solid and in solution is readily oxidized in air and inactivated by UV light.
- To reduce photodestruction of RE, manipulations of RE solutions can be performed under yellow or red light.
- Solutions may be stabilized by dissolving in oil, by the addition of anti-oxidant compounds including a-tocopherol or hydroquinone or by conversion to the palmitate and acetate esters.
- It is recommended to prepare solutions fresh for optimal quality. However, if absolutely necessary, store solutions in the dark under an inert atmosphere at least at -20°C preferably at -70°C
- Solvents preferred for storage are peroxide-free ethyl ether, acid-free acetone or ethyl acetate.
- For short term storage, ethanol is suitable as a solvent for spectroscopic analysis.
USAGE / APPLICATION
- The isolation of retinol from human plasma has been described.
- RE is an effective antioxidant displaying lipoperoxy radical scavenging activity.
- The interactions between RE and Vitamin E (a-tocopherol) in suppressing lipid peroxidation were observed in bovine retinal membrane preparations.
- RE may influence the production of transition vesicles by stimulating the activity of a protein disulfide isomerase-like activity involved in vesicle formation.
- RE may be involved in immune system mechanisms; an RE deficiency will depress the immune response producing a negative effect on both humoral and cellular immunity.
- (10 μM) and other retinoid compounds effectively induced sanguinarine and chelerythrine (benzophenanthridine alkaloids) accumulation in suspension-cell cultures of Sanguinaria canadensis in a way similar to fungal elicitation.
- RE (10 μM) stimulated DNA synthesis and possibly repair mechanisms in Sertoli cells of rat.
GENERAL NOTES
The USP unit of vitamin A (same as the International Unit6) is equal to 0.3 μg of the pure all-trans isomer of retinol which is equivalent to 0.344 μg of all-trans retinyl acetate. RE and its metabolites, including retinoic acid, are part of the retinoid class of compounds, involved in vision, normal embryo morphogenesis and in the regulation of proliferation and differentiation of a number of cell types. Current information and hypotheses on the absorption, transport, storage and metabolism of this fat soluble Vitamin A (retinol) have been reviewed. Studies on RE metabolism including its mobilization and transport in plasma and in tissues via serum and cytosolic retinol-binding proteins have been described.
STLC
STLC kdorfman Mon, 04/23/2018 - 17:51- STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47
(+)-S-Trityl-L-cysteine is a cell-permeable selective inhibitor of mitotic kinesin Eg5 and ATPase activities.
KIF11 (also known as kinesin-5 and Eg5) is a homotetramer which cross-links anti-parallel microtubules in the mitotic spindle to maintain spindle bipolarity. The motor domain or motor head is at the N-terminus and performs ATP hydrolysis and binds to microtubules. (https://en.wikipedia.org/wiki/Kinesin_family_member_11)
Make 100 mM stock:
- 1 g in bottle.
- Mix with 27.5 mL DMSO
- heat to 65C (~10 min), vortex
- if necessary, filter sterilize to remove insoluble particles.
- save as 1 mL aliquots in freezer. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)
Make 1 mM stock from the 100 mM stock
- 10 µL 1mM stock
- 990 µL DMSO
- vortex
- it will crystallize in the refrigerator. Warm (60C) and vortex to redissolve it. DO NOT LEAVE IN HOT BLOCK - IT WILL TURN BLACK!
Make 10 µL aliquots of 1 mM stock. Freeze
- label says to add 990 µL medium to make 10 µM working solution
Sodium alginate
Sodium alginate kdorfman Thu, 04/28/2022 - 14:52ALGINIC ACID SODIUM SALT 5G
Fisher 177770050 $22.41
(Can get 100g from Sigma for $55, but with long lead time)
(C6H7O7)A(C6H7O7)BNa
MW=120,000-190,000 g/moL
The following is from Sigma:
General description
Alginic acid sodium is a gelling and nontoxic anionic polysaccharide. The carboxylic acid groups on the alginic acid chain, renders it insoluble in water.However, converting alginic acid to its sodium form, enables it to solubilize in water easily.
Application
Alginic acid sodium is used:
- in combination with chitosan, to fabricate a biodegradable porous scaffold for bone tissue engineering.
- to study the characteristics of a modified amphiphilic alginate derivative
- to the study the impact of alginate on the rate of lipid digestion by employing an in vitro digestion model
- in the preparation of alginate hydrogels
- as encapsulating agents of microparticles of β-galactosidae
THC
THC kdorfman Fri, 04/22/2022 - 17:40Taxol
Taxol kdorfman Mon, 04/23/2018 - 17:51Taxol (20 µM final)
Fisher 109710 Paclitaxel.
Sigma T7402 1mg
MW = 853.91
- Stabilizes the microtubule polymer
- Protects microtulues from disassembly.
- Prevents metaphase spindle configuration
Blocks progression of mitosis and prolonged activation of the mitotic checkpoint triggers apoptosis or reversion to the G-phase of the cell cycle without cell division.
soluble in DMSO, not water.
Add 0.117 mL DMSO to make a 0.01M (=10 mM) stock solution from the 1 mg powder
Aliquot 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)
- add 490 µL to make 200µM
- dilute into medium from there (100 uL Taxol/mL medium)
Thyroid hormones
Thyroid hormones kdorfman Fri, 09/06/2019 - 18:38for Fish
5 mL per well for treatment
PTU (inhibitor) Sigma p3755
MW 170.2
Solubility:
- 50 mg/mL in 1M NaOH
- 16 mg/mL in alcohol
- 1 mg/mL in RT water
- 10 mg/mL in boiling water
In order to get the following doses, best to make it in hot fish water; otherwise, the NaOH or EtOH will be too concentrated in the final dilution.
dose 1: 0.5 mM
dose 2: 1.0mM
L-T4 (thyroxin) Sigma T2376
MW: 776.87
solubility:
- 50 mg/ml in 4M NH4OH in MeOH
- 0.5 mg/mL in 1:5 DMSO:PBS
Solution is good for ~ 1 day.
Make a 1 mM stock in 1M NaOH. Takes a long time. Vortex.
Priya's recipe: 1 mg/mL in 1M NaOH (~25.7 uM stock)
Working concentrations
dose 1: 100 nM (=0.1 uM)
dose 2: 300 nM (=0.3 uM)
Trichostatin A
Trichostatin A kdorfman Wed, 04/25/2018 - 19:52Cayman 89730
10 mM
Histone deacetylase Inhibitor
Trichostatin A (TSA), an antifungal antibiotic produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of histone deacetylases (HDACs), which are overexpressed in various cancers and closely correlate with oncogenic factors.
Trichostatin A is active at nanomolar concentrations in mammalian cells. By suppressing the activity of HDACs, it leads to increased histone acetylation, thereby causing highly acetylated histones to accumulate in the cell [2]. This in turn induces enhanced expression of specific genes that elicit extensive cellular morphologic and metabolic changes such as growth arrest, differentiation and apoptosis. At submicromolar concentrations Trichostatin A has been shown to induce apoptosis in diverse cancer cells while exhibiting very low toxicity to normal cells.
TSA is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, TSA should be directly dissolved in 0.1 M HCl (1.3 mg/ml) and then neutralized with PBS (pH 7.2) to achieve the desired concentration or pH. Approximately 0.7 ml of PBS (pH 7.2) is required to neutralize 1 ml of the acidic solution. We do not recommend storing the aqueous solution for more than one day.
Valinomycin
Valinomycin kdorfman Wed, 04/25/2018 - 21:03Potassium Ionophore, MW: 1111.32
Sigma V0627
Insoluble in water
stock is 10 mg/mL in DMSO
- cyclododecadepsi-peptide ionophore antibiotic.
- potassium ionophore that transports K+ across biological and artificial lipid membranes.
- can induce K conductivity in cell membranes
- uncouples oxidative phosphorylation,
- induces apoptosis in murine thymocytes, and in pre-B cell.
- inhibits NGF-induced neuronal differentiation
- antagonizes ET-induced vasoconstriction.
- Useful in studies of K transport in mitochondria.
Valproate
Valproate kdorfman Fri, 03/25/2022 - 15:34Sodium Valproate (Fisher 11474361)
Store powder at RT
Make 100 mM stock: 0.166g in 10 mL H2O
___ | ___ |
---|---|
SKU | 02152064-CF |
Alternate Names | 2-Propyl pentanoic acid; Valproic acid sodium salt; Sodium 2-propylpentanoate |
Application Notes | Sodium Valproate is reported to cause inositol depletion, activate the ERK pathway, inhibit GSK-3α and GSK-3β. Valproic Acid has been reported to be a potent inhibitor of HDAC (histone deacetylase) in vitro (IC50 = 400 μM for HDAC1), thereby relieving HDAC-dependent transcriptional repression and causes the hyperacetylation of histones in cultured cells. In animal studies, Valproic Acid has been observed to reduce tumor growth and metastasis formation. Additionally, Valproic Acid is reported to activate Wnt-dependent gene expression and to mimic trichostatin A in the inhibition of histone deacetylase. This compound is also an inhibitor of the CYP2C9 enzyme. |
Base Catalog Number | 152064 |
Biochemical Physiological Actions | Anti-convulsant that also has efficacy as a mood stabilizer in bipolar disorder. |
Boiling Point | 219.5 deg C |
CAS # | 1069-66-5 |
Density | 0.904 g/cu cm at 25 deg C |
EC Number | 213-961-8 |
Format | Crystalline powder |
Hazard Statements | H302 |
Molecular Formula | C8H15NaO2 |
Molecular Weight | 166.196 g/mol |
Personal Protective Equipment | Dust mask , Eyeshields, Gloves |
RTECS Number | YV7876000 |
Safety Symbol | GHS07 |
Solubility | In water, 2.0X10+3 mg/L at 20 deg C |
Usage Statement | Unless specified otherwise, MP Biomedical's products are for research or further manufacturing use only, not for direct human use. For more information, please contact our customer service department. |
Vapor Pressure | 8.47X10-2 mm Hg at 25 deg C (est) |
Vasopressin
Vasopressin kdorfman Mon, 04/23/2018 - 17:50(=Anti-diuretic hormone)
causes calcium oscillations
1 mM stock in water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)
1 mg in bottle, MW = 1084
Add 0.92 mL to make 1mM stock
1 µL aliquots in freezer
1 µM dilution to make student solutions
1 µL 1 mM in 999 µL water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)
Vitamin D
Vitamin D kdorfman Fri, 04/22/2022 - 17:53D3-calciferol
C27H44O
MW = 384.65
Solubility (from Cayman Chemical)
solvent | mg/mL | = mM |
---|---|---|
ethanol | 30 | ~78 mM |
DMSO | 3 | ~ 7.8 mM |
DMF | 25 | ~65 mM |
Stock solution: 29 mg/mL (~75 mM) in ethanol
General Description (from Thermo Scientific)
- Vitamin D3 is a steroid hormone generated in the skin when the 7-dehydrocholesterol interacts with ultraviolet irradiation. It can also be found in several types of food for daily intake.
- Vitamin D3 binds to vitamin D receptors, thus modulating gene expression
Applications
- In in vivo studies, vitamin D3 has been implicated in the maintenance of blood calcium and phosphorus levels, bone metabolism, metabolic functions, and transcription regulation
- It modulates the proliferation and differentiation of both normal and cancer cells
- It presents in vitro antiproliferative and antimetastatic activities on breast, colon, and prostate cancer cells
By target
By target kdorfman Wed, 06/20/2018 - 21:28Target or Effect | Drug(s) |
---|---|
Vasoconstriction | Angiotensin |
Non-muscle myosin II ATPase | Blebbistatin |
Internal calcium levels | Bradykinin |
Nervous system | Cannabinol |
α-Mannose, α-Galactose sugars, glycoproteins and glycolipids | Concanavalin A |
cyclin-dependent kinase | cyclin inhibitor |
actin polymerization | cytochalasin D |
cell adhesion | fibronectin |
Golgi/ER | brefeldin |
Primers
Primers kdorfman Fri, 05/31/2013 - 17:35Primers come lyophilized.
Tube label says how many nmol in the tube (usually ~100-500)
Multiply nmol x 10 = μL of sterile water to add to make 100 μM stock. Fisher primer calculator
This is the only stock!
Put it away safely on instructor shelf in freezer.
Make a working stock for students (give them all of it):
- 12.5 μM (12.5 μL of the 100 μM stock + 87.5 μL water).
(If using the repeater pipet for the water, 85 µL water + 12.14 µL 100 µM stock)
Sea Water
Sea Water kdorfman Thu, 02/16/2017 - 19:51Instant Ocean
- 34 g/L
- pulverize in mortar and pestle
- add very slowly to the water, stirring
- autoclaving helps the salts go into solution a little
Artificial Seawater, according to Wikipedia
salt | molarity |
---|---|
NaCl | 0.409 |
Na2SO4 | 0.003 |
KCl | 0.009 |
NaHCO3 | 0.0023 |
KBr | 0.00082 |
H3BO3 | 0.00042 |
NaFl | 0.00007 |
MgCl2 | 0.5327 |
CaCl2 | 0.01033 |
SrCl2 | 0.00009 |
Stains
Stains kdorfman Thu, 08/13/2015 - 20:47Acridine Orange
Acridine Orange kdorfman Fri, 07/27/2018 - 18:06Sigma A8097
10 mg/mL solution in water
MW = 308.81
Ex: 486 - 492
Em: 516 - 532
From the Sigma web page:
Application
Acridine Orange hydrochloride solution has been used to study autophagic cell death. It has also been used for the staining of chromosomes.
Biochem/physiol Actions
Acridine Orange is a metachromatic dye which can stain DNA, RNA and acid glycosaminoglycans. At low concentration it intercalates into DNA and precipitates RNA. However, at high concentration it denatures and precipitates both RNA and DNA. Acridine Orange is also used to analyze autophagy. It goes into acidic organelles in a pH-dependent manner. At neutral pH, acridine orange gives a green fluorescence and in acidic conditions, it accumulates in the acidic organelle giving a bright red fluorescence.
From use in MBoMS: Use at ~20µM
1.2 µL added to 2 mL in dish
incubate 15 min at 37
change medium
incubate 15 min at 37
Replace medium with PBS
observe with
- B excite - G emission: dsDNA
- G excite - R emission: RNA, ssDNA
Got good results initially, but within minutes, cells started to ball up and pull off the substrate. Will try new PBS first, then a concentration gradient of acridine orange.
Old PBS produced fast shrinking and balling up. Newer PBS was less bad. Now try with non-CO2 medium. Does it interfere with fluorescence?
No cell shrinkage in non-CO2 medium. At short exposure times, there is a background glow, but picking the right exposure takes care of that.
This is time sensitive. The red emission (which should be RNA & ssDNA) gradually fades, or overlies the green emmision (which should be ds DNA)
See attached images, taken in order 1, 2, 3. #3 is at about 15 minutes. 1 & 2 are 100x, 3 is 200x.
Click-iT® Plus EdU Imaging Kits
Click-iT® Plus EdU Imaging Kits kdorfman Mon, 08/12/2019 - 18:55Materials required but not in kit:
3% BSA in PBSTX
Stock Solutions
Component | conc | dilute with |
---|---|---|
A (EdU) | 10 mM | 2 mL DMSO (C) or buffer |
B (Alexa Fluor® picolyl azide) | ||
C (DMSO) | ||
D (reaction buffer) | 10X | |
E Cu protectant | ||
F (buffer additive) | ||
G (Hoechst® 33342) |
Per tube, need 200uL
Buffer Additive
Buffer Additive kdorfman Thu, 03/26/2020 - 14:18To make a 10X stock solution of the Click-iT® EdU buffer additive (Component F): Add 2 mL deionized water to the vial, then mix until fully dissolved. After use, store any remaining stock solution at ≤–20˚C.
When stored as directed, this stock solution is stable for up to 1 year. If the solution develops a brown color, it has degraded and should be discarded.
EdU
EdU kdorfman Wed, 09/11/2019 - 17:0033mM (10X) Stock solution
100 mg of EdU powder (purchased from Carbosynth) https://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/D1…
is dissolved in
1.2 mL DMSO, then brought to 12 mL with H2O
Original recipe says: 10.8mL 0.5x E2 medium and
1.2ml DMSO
Rolf says the final concentration of DMSO should be more like 0.1% (after dilution of the 10X EdU to 1X in fish water)
Says to mix with water and warm gently (to thaw, use 60C for 15 minutes)
So try 33 mM EdU in 1% DMSO.
To make 20 mL 10X EdU in 1% DMSO, mix
- 166 mg EdU
- 0.2 mL DMSO (heat gently by putting it in a beaker of heated water)
- bring to final volume with distilled water
Reaction Buffer
Reaction Buffer kdorfman Thu, 03/26/2020 - 14:13Prepare a working solution of 1X Click-iT® EdU reaction buffer (Component D):
Transfer the solution (4 mL) in the Component D bottle to 36 mL of deionized water.
To make smaller amounts of 1X Click-iT® EdU reaction buffer,
dilute volumes from the Component D bottle 1:10 with deionized water.
After use, store any remaining 1X solution at 2–8˚C.
When stored as directed, this 1X solution is stable for 6 months.
DAPI Fluoromount
DAPI Fluoromount kdorfman Fri, 03/31/2023 - 21:35DAPI Fluoromount Fisher OB010020
Store at room temp, in the dark
Aliquot 1 mL into dark tube.
Keep in "mounting" drawer in 360.
Make droppers by melting the end of a glass Pasteur pipet to make a ball.
DAPI in glycerol for fish
DAPI in glycerol for fish kdorfman Thu, 02/24/2022 - 19:361 mg/mL DAPI stock solution from Rolf
1 uL per 10 mL 25% glycerol
DNA gel stains
DNA gel stains kdorfman Mon, 08/27/2018 - 20:573-color LD + SYBR Safe
3-color LD + SYBR Safe kdorfman Mon, 10/15/2018 - 17:38The orange runs fast; suitable for small DNA fragments
To make 10 mL from home made LD:
- 6 mL orange G (Fisher AAJ60562AC)
- 1 mL home made LD with bromophenol blue and xylene cyanacol
- 6 uL SYBR safe
- 1 mL glycerol
- 0.1 mL 1 M Tris pH 7.6
- water to 10 mL
EtBr
EtBr kdorfman Mon, 08/27/2018 - 21:01Ethidium bromide
1 uL / 100 mL gel (or DNA sample, if adding it to the loading dye) (=1/100,000)
10 mg/mL
10 mL
17-ETBC1001 Krackeler $37.12
Home-made
Home-made kdorfman Mon, 08/27/2018 - 21:136X Home-made loading dye:
- 30% glycerol
- 0.3% Bromphenol blue
- 0.3% xylene cyanol
plus SYBR-Safe at 0.5 uL/1mL
1 uL loading dye stains 5 uL of DNA sample.
SYBR safe works at 1/10,000
need 5/10,000 uL for 5 uL of sample
so need 5/10,000 uL SYBR safe for each uL of loading dye.
= 5/10 uL for each mL
NOTE Fisher Tritrack loading dye (FERR1161) composition:
- 0.03% bromophenol blue
- 0.03% xylene cyanol
- 0.15% orange g
- 60% glycerol
- 10 mM Tris, pH 7.6
- 60 mM EDTA
One tenth as much dye, twice as much glycerol.
SYBR Safe
SYBR Safe kdorfman Mon, 08/27/2018 - 21:04SYBR safe DNA gel stain
Fisher S33102
400 uL $72.62
Use 1 uL/10 mL gel or DNA sample (=1/10,000)
Safe-Green
Safe-Green kdorfman Mon, 08/27/2018 - 21:13Safe-Green
(DNA stain + loading dye)
ABM G108-G
1 mL $65.00 ($30 + shipping)
Use at 1:5
Smart-glow
Smart-glow kdorfman Mon, 08/27/2018 - 21:07Smart Glow loading dye
Krackeler 26510-E4500-LD
1mL $77.22
DNA stain + loading dye.
Add directly to DNA sample
Use at 1:5
DiOC6(3)
DiOC6(3) kdorfman Fri, 05/11/2018 - 16:44Invitrogen D273
DiOC6(3) is a cell-permeant, green-fluorescent, lipophilic dye that is selective for the **mitochondria:: of live cells, when used at low concentrations. At higher concentrations, the dye may be used to stain other internal membranes, such as the endoplasmic reticulum.
ER Tracker
ER Tracker kdorfman Thu, 04/05/2018 - 15:34ER Tracker Red
(BODIPY™ TR Glibenclamide), for live-cell imaging Invitrogen/Life/ ThermoFisher: E34250
Ex = 587 nm Em = 615 nm
MW = 915.23
100 ug lyophilized.
To make stock solution:
- Add 110 uL DMSO to make a 1mM solution
- Make 1 uL aliquots
To use
Working concentration: ~1 uM.
Fluo-4 AM
Fluo-4 AM kdorfman Thu, 04/26/2018 - 18:04Fluo-4 stock
Invitrogen F14217 500 µL
Calcium indicator (fluoresces when bound to Calcium ions)
Ex 494 nm; Em 516 nm
1mM in DMSO
Protect from light
Store in dissicator.
20 µL aliquots. Each makes 10 mL of 2 µM solution
Fluo-4 staining solution
2 µM Fluo-4 + 0.02% pluronic in HBS
Incubate 15 - 60 min at 20 - 37C. Wash before viewing.
plus HBS to final volume
Lysotracker
Lysotracker kdorfman Thu, 10/24/2019 - 20:17Lysotracker (Invitrogen L-7528 - 20 x 50 µL)
50 - 75 nM
stock = 1 mM in DMSO
aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
30 min - 2 hours incubation warm.
Mitotracker
Mitotracker kdorfman Wed, 02/21/2018 - 18:59Mitotracker GREEN
Mitotracker GREEN kdorfman Thu, 10/24/2019 - 20:34Excitation 490 nm : Emission 516 nm
MW: 674
50 ug in vial.
Add 74 uL DMSO to make 1 mM stock.
working concentration =~ 25 - 200 nM
1 uL 1mM stock into 10 mL medium makes 100 nM treatment solution
Mitotracker RED
Mitotracker RED kdorfman Thu, 10/24/2019 - 20:34Invitrogen M7512
https://www.thermofisher.com/order/catalog/product/M7513?SID=srch-srp-M…
Ex = 579 nm; Em = 599
50 ug per tube.
MW = 531
Add 100 uL DMSO to 50 ug in tube. Makes ~ 1 mM stock solution.
Use at 25 - 100 nM. (1 uL per 10 mL for 100 nM)
Dilute in medium. Treat with pre-warmed stain solution for 15 - 45 minutes. Replace staining solution with fresh (warm) medium.
To fix: use 3.7% formaldehyde (in medium); 37C for 15 minutes (??)
NBD Ceramide
NBD Ceramide kdorfman Wed, 04/25/2018 - 14:32Invitrogen N22651
fluorescent marker for Golgi in live cells
Ex: 466nm Em: 536nm
Follow instructions attached: Add 150 µL sterile H2O to the 5 mg in the bottle. (Makes 0.5mM (=500 µM) in BSA)
Aliquot 10 µL, and freeze
Working concentration is 5 µM
Add 990 µL HBSS (Hank's)(0.34 mg/mL BSA) to 10 µL in tube
Makes enough for 8 groups.
Aliquot ~120 µL per group, enough to cover the small circular coverslip in the viewing dish.
To use:
- Rinse with HBSS (see notes)
- Treat with ceramide solution
- incubate in cold 30 min
- rinse 3x in cold HBSS
- replace HBSS with non-CO2 medium, incubate ~20 minutes
NOTES:
Minimal rinse! Very gently! Cold cells will fall off!
If following with ER tracker, rinse with HBSS, then replace with warm non-CO2 medium
If following with Mitotracker, rinse with PBS, then replace with warm non-CO2 medium
Nuc Blue
Nuc Blue kdorfman Thu, 07/14/2022 - 18:02Hoechst type DNA stain for live cells
Nuc Blue protocol from Thermo Fisher
- Culture cells in an appropriate medium and vessel for fluorescence microscopy.
- Add two drops (20 uL = 1 drop) of NucBlue Live ReadyProbes Reagent per milliliter of medium.1
- Incubate for 20 minutes, protected from light.
- Image the cells.
-
In some cases, more or fewer drops may be needed to achieve optimal staining intensity. Image quality may be improved by replacing the culture medium with Live Cell Imaging Solution (Cat. No. A14291DJ). [Kate says start with less, and work up. Rinse cells after incubation.] ↩︎
Nucleus-RFP
Nucleus-RFP kdorfman Fri, 10/27/2023 - 18:57CellLightTM Nucleus-RFP
Invitrogen (Thermo-Fisher) C10603
1 mL - received 10/26/23 - purchased for Bioimaging
Store in refrigerator
Simplified protocol:
Starting concentration = 108 particles per mL
Working concentration = 10 - 50 particles per cell
Use low-passage number cells;
estimate number of cells at time of treatment - should be no more than 70% confluent
(number of cells x ~30 particles/cell)/108 particles/mL = mL CellLight to use
Mix thoroughly but gently with cell medium
Image cells after about 16 hours
From the mfgr:
CellLight Nucleus-RFP, BacMam 2.0, provides an easy way to label nuclei with red fluorescent protein (RFP) in live cells. Simply add the reagent to your cells, incubate overnight, and the cells are ready to image in the morning.
This ready-to-use construct is transfected into cells using BacMam 2.0 technology, where it expresses RFP fused to the SV40 nuclear localization sequence. You can observe nucleus-RFP behavior in live cells without the cellular toxicity associated with intercalators and label with multiple tracking or tracing dyes to image dynamic cellular processes.
Cells expressing CellLight constructs can also be fixed with formaldehyde for multiplexed imaging using immunocytochemical techniques.
CellLight Technology is:
- Fast and convenient: simply add CellLight reagent to your cells, incubate overnight, and image—or store frozen, assay-ready cells for later use
- Highly efficient: up to 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
- Flexible: co-transduce more than one BacMam reagent for multiplex experiments or co-localization studies; tightly control expression levels by simply varying the dose
- Less toxic: CellLight reagents are non-replicating in mammalian cells and are suitable for biosafety level (BSL) 1 handling
BacMam Technology
CellLight Nucleus-RFP, BacMam 2.0, is a fusion construct of SV40 nuclear localization sequence and TagRFP, providing accurate and specific targeting to cellular nucleus-RFP. This fusion construct is packaged in the insect virus baculovirus, which does not replicate in human cells and is designated as safe to use with biosafety level (BSL) 1 in most laboratories. BacMam technology ensures that most mammalian cell types are transduced/transfected with high efficiency and minimal toxicity. This transient transfection can be detected after overnight incubation for up to five days—enough time to carry out most dynamic cellular analyses. Like any transfection/transduction technique, the BacMam method does not transfect/transduce all of the cells with equal efficiency, making it poorly suited to cellular population studies or automated imaging/counting. CellLight reagents are ideal for experiments where cellular or subcellular co-locatization is required, or for cellular function studies that need special resolution.
Visualize staining your cell without wasting your reagents, antibodies, or time with our new Stain-iT Cell Staining Simulator.
PS-Speck
PS-Speck kdorfman Thu, 02/01/2018 - 13:43Fluorescent Beads
(Should also try MultiSpeck beads M 7901)
Protocol:
- use polylysine slides so beads stick
- 5 uL beads total (can mix colors on one slide)
- 12 uL water; mix well
- dry on a slide warmer (or hot plate at about 50C)
- draw a circle on the bottom of the slide around the dried material
- put 1 drop mounting medium over dried spot (gloppy! can't pipet!)
- coverslip
- nail polish
Molecular Probes P7220
Code | Color | Ex (nm) | Em (nm) | filter cube label |
---|---|---|---|---|
A | blue | 360 | 440 | UV |
B | green | 505 | 515 | B |
C | orange | 540 | 560 | LP (?) G? |
D | deep red | 633 | 660 | probably not |
Phalloidin
Phalloidin kdorfman Thu, 08/13/2015 - 21:03General protocol:
Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)
Aliquots
- 5 µL stock per 0.5 mL tube
- Label: Add 200 µL PBS (or PBS 1% BSA), use 50µL per coverslip
Fix cells with formaldehyde
Procedure:
- 50 uL phalloidin on parafilm in humid chamber
- coverslip cell side down onto drop
- 15 - 20 minutes at room temp
- rinse repeatedly in PBS-Tween-azide
- blot the corner on a kimwipe
- Mount on a drop of mounting medium on a slide
FITC phalloidin
FITC phalloidin kdorfman Fri, 06/01/2018 - 18:40Thermo Fisher F432
From the manufacturer:
Fluorescein phalloidin is a high-affinity F-actin probe conjugated to the green fluorescent dye, fluorescein (FITC).
- Selectively stains F-actin
- Excitation/Emission: 496/516 nm
- Superior to antibody staining
- Optimal for fixed and permeabilized samples
10 and 20 uL aliquots
In histology freezer
Red phalloidins
Red phalloidins kdorfman Fri, 06/01/2018 - 18:42Alexa Fluor 568 Phalloidin
Alexa Fluor 568 Phalloidin kdorfman Fri, 06/01/2018 - 18:41Alexa Fluor 568 Phalloidin
Fisher or Invitrogen A 12380
Binds to F-Actin
Ex/Em 578/600
Stock Solution: Add 1.5 mL MeOH to vial (~6.6µM)
Aliquots
- 8 µL stock per 0.5 mL tube
- Label: Add 400 µL PBS (or PBS 1% BSA), use 50µL per coverslip
Fix cells with formaldehyde
Procedure:
- 50 uL phalloidin on parafilm in humid chamber
- coverslip cell side down onto drop
- 15 - 20 minutes at room temp
- rinse repeatedly in PBS-Tween-azide
- blot the corner on a kimwipe
- Mount on a drop of mounting medium on a slide
In histology and bioimaging freezers
Rhodamine Phalloidin
Rhodamine Phalloidin kdorfman Fri, 06/01/2018 - 18:35Thermo Fisher R415
From the manufacturer:
Rhodamine phalloidin is a high-affinity F-actin probe conjugated to the red-orange fluorescent dye, tetramethylrhodamine (TRITC).
- Selectively stains F-actin
- Excitiation/Emission: 540/565 nm
- Superior to antibody staining
- Optimal for fixed and permeabilized samples
- Very widely cited fluorescent phalloidin conjugate
In 262A freezer
Texas Red-X Phalloidin
Texas Red-X Phalloidin kdorfman Fri, 06/01/2018 - 18:30Thermo Fisher T7471
From the manufacturer:
Texas Red®-X phalloidin is a high-affinity F-actin probe conjugated to our bright, photostable, red fluorescent Texas Red®-X dye.
- Selectively stains F-actin
- Outstanding fluorescence performance
- Excitation/Emission: 591/608 nm
- Superior to antibody staining
- Optimal for fixed and permeabilized samples
In histology freezer
Phloroglucinol
Phloroglucinol kdorfman Wed, 03/17/2021 - 16:10Stains lignin
Phloroglucinol-HCl (Wiesner) Staining
Dissolve 0.3 g of phloroglucinol in 10 mL absolute ethanol to prepare a 3% phloroglucinol solution.
Mix one volume of concentrated HCl (37 N) to two volumes of 3% phloroglucinol in ethanol; this solution is phloroglucinol-HCl (Ph-HCl) or Wiesner stain. May 13, 2014
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186213/
3% Phloroglucinol solution
Ph-HCL stain
Syto RNASelect
Syto RNASelect kdorfman Wed, 04/25/2018 - 19:47SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)
$210 from Invitrogen
Fluoresces green when bound to RNA.
excite: 490 nm, emit: 530 nm
Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!
Don't use in conjunction with red-orange dyes.
Stock:
100µL 5 mM in DMSO. Store at -20C, dessicated, dark.
To thaw: warm to RT, spin down.
Should be stable for >= 1 year.
Make labeling solution
Make 5µM intermediate stock:
2 µL 5mM stock + 1998 µL medium or PBS
Make 20 100 µL aliquots in 1.5 mL tubes
Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution
Make 500 nM labeling solution in medium or PBS
100 µL 5 µM intermediate + 900 µL medium
Protocol
Live cells
- Cells on coverslip
- Warm 500 nM labeling solution to 37C
- Incubate at 37C 20 min
- Rinse twice in PBS or medium
- Add warm medium, let cells rest 5 min
- Fix in chilled methanol 10 min at -20C
- Several washes in PBS
Fixed cells
- Remove coverslip from medium
- Fix in chilled methanol 10 min at -20C
- Remove methanol, let slip sit in PBS 5 min
- Apply labeling solution 20 min RT
- Wash 5 min in PBS
- Mount coverslip
TMRE
TMRE kdorfman Wed, 04/25/2018 - 21:17Tetramethrylrhodamine ethyl ester perchlorate
mitochondria-specific Red fluophore
Biochemika (Sigma) 87917
Soluble in DMSO, alcohols.
Stock is 50 mM in DMSO
λex 540 nm; λem 595 nm in DMSO
cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria.
Potential-sensitive probe for measuring membrane potential changes in mitochondria
In freezers in 266A and 362A
Tetramethylrhodamine α-Bungarotoxin
Tetramethylrhodamine α-Bungarotoxin kdorfman Fri, 06/01/2018 - 19:06Thermo Fisher T1175
Binds to acetylcholine receptor at neuromuscular junction
From the manufacturer:
Tetramethylrhodamine a-bungarotoxin can be used to visualize this receptor. Labeled a -bungarotoxin conjugates can be used to facilitate identification of nicotinic AChRs and to localize neuromuscular junctions.
Excitation⁄Emission (nm): 554⁄577
In freezer 266A
Toluidine blue
Toluidine blue kdorfman Wed, 03/17/2021 - 16:12Toluidine blue is a basic thiazine metachromatic dye with high affinity for acidic tissue components.
It stains nucleic acids blue and polysaccharides purple and also increases the sharpness of histology slide images. It is especially useful today for staining chromosomes in plant or animal tissues
Toluidine blue for Madelaine's plant anatomy class
- 1 % Toluidine blue
- 0.5% borax
Toluidine Blue Stock Solution:
1g in 100 mL 70% ethanol
NaCl acid solution
1% NaCl aqueous, pH between 2 - 2.5 (w/ glacial acetic acid)
Working toluidine solution
1 part toluidine stock solution : 9 parts NaCl-Acid
Make this solution fresh and discard after use. pH higher than 2.5 will make staining less contrast.
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&ved=2ahU…
Transferrin
Transferrin kdorfman Thu, 05/10/2018 - 14:49Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM).
Final concentration = 1 µM.
Aliquot 2.5 µL.
Add 150 µL Fe-HBS-BSA to make 153 µL 1 µM
Store in freezer
Transferrin is a monomeric serum glycoprotein (~80,000 daltons) that binds up to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis.
Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of iron from the transferrin–receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to scavenge more iron.
Fluorescent transferrin conjugates can therefore be used with fluorescent LDL to distinguish the lysosomally directed and recycling endosomal pathways.
Xylene Cyanol
Xylene Cyanol margaret Fri, 10/14/2011 - 15:59Stock solutions
Stock solutions kdorfman Fri, 10/28/2011 - 15:35ABA
ABA kdorfman Tue, 11/17/2015 - 19:28(+)-cis,trans-abscisic acid
Plant Media 30631017-1 250 mg $68
soluble in EtOH, MeOH, DMSO 20 - 50 mg/mL
MW = 264.3
Store as powder in freezer.
Make 1 mL of 100mM solution:
0.1mol/1000mL x 164.3g/mol = 0.0264 g
Working concentration ~0.15 mM
In 30 mL MS agar, =
vi = 30mL x 0.15 mM/100 mM = 0.045 mL
ATP
ATP kdorfman Mon, 11/04/2013 - 17:0210 mM ATP Stock
(from Maniotis)
60 mg ATP in 8 mL H2O
pH to 7.0 with 0.1 M NaOH
Bring volume to 10 mL with H2O
Aliquot and freeze.
Use at ~10 µM
Adenine
Adenine kdorfman Wed, 10/18/2017 - 22:41Fisher AC147440250 25 gr.
1 mg/mL solution for yeast MV-Ade medium
heat (65C) and stir for one & a half hours
filter sterilize
Adenine hemi-sulfate
Adenine hemi-sulfate kdorfman Wed, 01/15/2020 - 21:3910X Adenine hemi-sulfate (Sunrise ScienceProducts 1905-010)
2 g/L
Dissolve 2 g of Adenine per 1 L distilled water and filter sterilize. Store in the dark at 4 ˚C.
Antibiotics
Antibiotics kdorfman Wed, 12/28/2011 - 21:01Amphotericin
Amphotericin kdorfman Tue, 01/09/2018 - 18:29Anti-Fungal
Stock solution: 10 mg/mL in DMSO (insoluble in ethanol)
Store at 4C short term, -20 long term (in 15 mL tube, wrapped in foil with tape label)
Keep in the dark.
Final concentration in medium = 10 ug/mL
Add 1 mL per L
Freezes in the refrigerator.
Protect from light.
Ampicillin
Ampicillin kdorfman Wed, 12/28/2011 - 21:0250 mg/mL stock
(Final concentration in medium = 50 µg/mL)
Lasts up to 4 weeks in poured agar at 4C
0.5 g in 10 mL water
filter sterilize
1 mL aliquots (enough for 1 L medium)
0.4 mg/mL for QSB
Make 35 mL: 0.28 mL 50 mg/mL stock
35 mL sterile water
(filter sterilize if any doubt about sterility)
48 0.7 mL aliquots
Can also use carbenicillin
Carbenicillin
Carbenicillin kdorfman Tue, 08/02/2016 - 15:05(More stable than ampicillin)
Fisher 50841234b 25 mL Teknova C2130
100 mg/mL stock
effective concentration 50 - 100 µg/mL in medium
Freeze aliquots.
Chloramphenicol
Chloramphenicol kdorfman Thu, 04/23/2015 - 13:37Cipro
Cipro kdorfman Wed, 12/28/2011 - 21:02Ciprofloxacin 10 mg/mL stock
(final concentration in medium = 10 µg/mL)
100 mg in 10 mL dilute acid (add HCl drop by drop, mixing in between till it dissolves)
filter sterilize
1 mL aliquots
freeze
0.2 mg/mL for QSB
0.7 mL 10 mg/mL stock
35 mL water
filter sterilize
48 0.7 mL aliquots
freeze
Doxycycline
Doxycycline kdorfman Mon, 01/18/2016 - 13:27Tetracycline category
solubility 50 mg/mL in water
disk has 30 ug
Erythromycin
Erythromycin kdorfman Fri, 01/20/2012 - 18:10solubility
50 mg/mL in ethanol
lasts up to 4 weeks in poured agar at 4C
10 mg/mL stock
(10 µg/mL in medium)
0.25 g in 25 mL EtOH
1 mL aliquots
freeze
0.6 mg/mL for QSB
2.1 mL 10 mg/mL stock
33 mL EtOH1
48 0.7 mL aliquots
freeze
-
Try 20 µL in 330 µL water to see if it dissolves. If so, then make aqueous solution, filter sterilize, and freeze. ↩︎
Kanamycin
Kanamycin kdorfman Wed, 12/28/2011 - 21:0150 mg/mL stock (50 µg/mL in medium)
Concentration in poured agar drops to ~80% in 1 week, but stays stable for 3 more weeks at 4C
1.25 g in 25 mL H2O
filter sterilize
mL aliquots
freeze
1.2 mg/mL for QSB
0.84 mL 50 mg/mL stock
34.16 mL water
filter sterilize (if any doubt about sterility)
48 0.7 mL aliquots
freeze
Kan calcs
Kan calcs kdorfman Tue, 10/02/2012 - 16:23Streptomycin
Streptomycin kdorfman Tue, 10/17/2023 - 19:43In refrigerator in 362A
Fisher Streptomycin sulfate 50g
BP910-50 (discontinued)
MW 1457.58
~50 mg/L for agar
Tetracycline
Tetracycline kdorfman Wed, 01/18/2012 - 20:28Standard is 10 mg/mL stock
Stable for 2 weeks in poured agar at 4C; 75% after 4 weeks
15 mg/mL stock
0.275 g tetracycline in 25 mL EtOH
1 mL aliquots
freeze
1.2 mg/mL for QSB
2.8 mL 15 mg/mL stock in 32.2 mL EtOH1
48 0.7 mL aliquots
freeze
-
Try 28 µL in 322 µL water to see if it dissolves. If so, then make aqueous solution, filter sterilize, and freeze. ↩︎
Triclosan
Triclosan kdorfman Wed, 12/28/2011 - 21:02Irgasan
Sigma 72779-5g-f
MW 289.54
Boric acid
Boric acid kdorfman Tue, 12/20/2011 - 15:09Boric acid (H3BO3) 0.01 M (=10 mM)
1000x for M&S micronutrients
MW = 61.83
0.031 g/50 mL
Calcium chloride
Calcium chloride kdorfman Tue, 12/20/2011 - 15:01CaCl stocks in two concentrations:
MW (dihydrate) = 147
2.205 g/15 mL
7.35 g/50 mL
**Autoclave (or otherwise sterilize) for *C. elegans* medium**
1 Molar
1.1 g/15 mL
3.675 g/50 mL
**Autoclave (or otherwise sterilize) for *C. elegans* medium**
0.5 Molar (=500 mM)
Calcium nitrate
Calcium nitrate kdorfman Mon, 08/03/2015 - 18:47Ca(NO3)2.4H2O
FW = 236.1
0.4M stock:
94.4g/L = 0.189g/50mL
Chelated Iron
Chelated Iron kdorfman Fri, 01/12/2024 - 21:28FeSO4.7H2O 2.78 g/L
Disodium EDTA 3.73 g/L
Mix each component in 450 mL water
Heat EDTA to boiling
Add EDTA to the FeSO4 solution.
Boil for an hour, cool completely
Bring to final volume
Cholesterol
Cholesterol kdorfman Mon, 01/09/2012 - 17:475 mg/mL for C. elegans medium
Fisher AAA1147018 Alfa Aesar 50 g ~$35
in 95% ethanol
Stir for 3 hours
or vortex several minutes
Do not autoclave!
Cobalt Chloride
Cobalt Chloride kdorfman Tue, 12/20/2011 - 15:11Cobalt Chloride (CoCl2) 105 µM (=~ 0.1 mM)
10,000X for M&S micronutrients
MW (hexahydrate) = 237.93
0.00125 g/50 mL
DNA standards
DNA standards kdorfman Wed, 12/22/2021 - 21:38NEB MARKERS
NEB DNA standards (for general reference)
NEB 1 Kb ladder (we have quick load and regular)
FISHER MARKERS. (See attached pdfs below for pictures of ladders)
Thermofisher DNA ladders (for general reference)
Gene ruler ladders (for general reference)
Fisher Scientific molecular weight markers (for general reference)
DTT
DTT kdorfman Fri, 05/31/2013 - 17:21Dithiothreitol
Acros organics426380500 (store refrigerated in 362A) $98 at Fisher
154.25 MW
1M = 1.5425g in 10 mL H20
EDTA
EDTA kdorfman Tue, 12/20/2011 - 14:45EDTA (disodium) 0.5 M pH 8.0
MW = 372.4
18.62 g/100 mL
9.31 g/50 mL
initial pH = ~6. Use NaOH pellets to bring to pH = 8. ~2 g NaOH/100 mL
EGTA
EGTA kdorfman Fri, 08/09/2013 - 20:27EGTA 0.5M
100 ml solution
19g EGTA (MW 380g/mol) ddH2O to 90ml adjust pH 7.5/8.0 with solid NaOH (>4g) adjust volume to 100ml
Note: EGTA will not go into solution without NaOH. Once the pH has been raised sufficiently it dissolves quickly. For pH 7.5 the exact amount required is slightly above 4g. Add 3.5-4g immediately, then proceed carefully not to overshoot the desired pH.
from http://www.researchgate.net/post/How_can_I_dissolve_EGTA
Ferric citrate
Ferric citrate kdorfman Tue, 12/20/2011 - 15:14Ferric citrate (C6H5FeO7) 89.4 mM
MW=244.95
1.095 g/50 mL
0.328 g/15 mL
Keep refrigerated
The 89.4 value is because Tobias Baskin's lab makes a 89.4 µM Fe-citrate medium, and it's easy to mix up a batch from the 1000x stock solution.
Maximum solubility is 1 g/100 mL hot H2O
Could in the future make a 10 mM batch with 0.1225 g in 50 mL, and for this lab: https://wahoo.nsm.umass.edu/content/reagents-61 , you would have easier calculations.
Ferrous sulfate
Ferrous sulfate kdorfman Tue, 12/20/2011 - 15:16Ferrous sulfate (FeSO4) 10 mM
MW (heptahydrate) = 278.01
MS 10x = 28mg/L = 0.1 mM - 100 µM
MS 1x = 0.01 mM = 10 µM
make 1,000x for M&S medium = 0.01 M = 10 mM
Keep refrigerated
Turns yellow. Probably oxidation. Yellow comes off on a micropore filter.
Fluorescein
Fluorescein kdorfman Mon, 10/29/2018 - 17:44MW = 332.31
Excitation wavelength: 460 nm Emission wavelength: 515 nm
Absorption maxima at 493.5 and 460 nm
A = Ecl
Molar extinction coefficient of fluorescein at 485nm = 50358/mol.cm
so Absorbance 10^-5M = 0.50358
(1/100 of a 1mM solution =10^-5M)
1 mM stock in 10mM NaOH
Histidine HCl
Histidine HCl kdorfman Wed, 01/15/2020 - 21:41100X Histidine-hydrochloride
8.56 g/L
Dissolve 2.14 g Histidine in 250 mL distilled water and filter sterilize.
Store in the dark at 4 ˚C.
IPTG
IPTG kdorfman Tue, 12/11/2018 - 20:471 M IPTG Stock Solution (isopropyl Beta-D-thioglucopyranoside, MW 238.3 g/mol)
2.383 g IPTG in 10 mL ddH2O.
Filter sterilize with 0.22 um membrane cartridge.
or buy 25 mL 1M stock solution Teknova 13431 (Fisher)
Store at -20C
Leucine
Leucine kdorfman Wed, 01/15/2020 - 21:4310X Leucine
1.8 g/L
Dissolve 0.18 g Leucine in 100 mL distilled water and filter sterilize.
Store in the dark at 4 ˚C.
LiOAc-SDS
LiOAc-SDS kdorfman Mon, 07/31/2023 - 21:51LiOAc-SDS for DNA extraction from yeast
To make 100 mL of
Mix 20 mL LiOAc
with 5 mL SDS
and bring to final volume
and bring to final volume
Lithium Acetate
Lithium Acetate kdorfman Wed, 01/15/2020 - 21:26Lithium acetate dihydrate
C2H7LiO4
Fisher AA1341730
MW = 102.014
adjust pH to 7.5 with dilute acetic acid for Laney's cell & Molec lab
filter sterilize
store at room temp
Lithium Chloride
Lithium Chloride kdorfman Mon, 03/12/2012 - 14:28LiCl 6M
MW = 42.39
Gets hot!!
MES
MES kdorfman Sun, 09/11/2016 - 17:47MW = 195.2
150 mM stock solution for LPGM
Magnesium chloride
Magnesium chloride kdorfman Tue, 12/20/2011 - 15:05Magnesium chloride (MgCl2) 1M
MW (hexahydrate) = 203.31
10.166 g/50 mL
20.33 g/100 mL
For Marine Biology (424) anesthetic reagent
for Aiptasia, make 0.37M in 30 - 35 PSU seawater
for Nematostella, dilute again 1:1
Magnesium sulfate
Magnesium sulfate kdorfman Tue, 12/20/2011 - 15:06Magnesium sulfate (MgSO4) 1M
MW (heptahydrate) = 246.48
Manganese chloride
Manganese chloride kdorfman Thu, 01/19/2012 - 07:30MnCl2 4H2O
M.W.197.9
5 mM
0.0495g/50mL
Manganese sulfate
Manganese sulfate kdorfman Tue, 12/20/2011 - 18:03MnSO4
MW (monohydrate) = 169
store dry in dessicator
MS 10X micronutrients: 16.9 mg/L = 0.1 mM
so 1X = 0.01mM = 10 µM
1000X MS = 10 mM
Mannitol
Mannitol kdorfman Tue, 08/12/2014 - 17:45MW = 182.172
Potassium acetate
Potassium acetate kdorfman Tue, 12/20/2011 - 15:15Potassium acetate (C2H3O2K)
Fisher BP364-500 $46.30
MW = 98.14
49 g in 100 mL H2O
Cloudy. Heat? Stir overnight? Lower the pH?
Can buy 5M solution: RICCA R5866500-1A ~$90 for 1L from Fisher
Potassium chloride
Potassium chloride kdorfman Tue, 12/20/2011 - 15:04Potassium chloride (KCl)
MW = 74.56
Potassium ferricyanide
Potassium ferricyanide kdorfman Tue, 12/20/2011 - 15:13Potassium ferricyanide (K3Fe(CN)6) 50 mM (= 0.05 M)
red salt
MW = 329.26
0.8232 g/50 mL
Store frozen
Potassium ferrocyanide
Potassium ferrocyanide kdorfman Tue, 12/20/2011 - 15:12Potassium ferrocyanide (K4Fe(CN)6) 50 mM (= 0.05M)
yellow salt
MW (trihydride) = 422.41
Store frozen
Potassium hydroxide
Potassium hydroxide kdorfman Thu, 01/19/2012 - 16:33KOH
MW = 56.11
Use for adjusting PIPES pH
Potassium nitrate
Potassium nitrate kdorfman Tue, 12/20/2011 - 15:18Potassium nitrate (KNO3)
Store in refrigerator
Potassium phosphate dibasic
Potassium phosphate dibasic kdorfman Sat, 01/14/2012 - 05:06K2HPO4
MW = 174.18
Potassium phosphate monobasic
Potassium phosphate monobasic kdorfman Tue, 12/20/2011 - 14:58Potassium phosphate monobasic (KH2PO4)
for C. elegans medium:
pH to 6.0 with solid KOH
(~1.7 g/100 mL)
Sterilize (can be autoclaved)
SDS
SDS kdorfman Tue, 12/20/2011 - 14:40SDS 20%
Sodium dodecanesulfate (=Sodium lauryl sulfate, NOT laureth)
CH3(CH2)11OSO3Na
DO NOT AUTOCLAVE - it (like all detergents) can boil over
Sodium acetate
Sodium acetate kdorfman Tue, 12/20/2011 - 15:08Sodium acetate (NaOAc) 3M
CH3COONa
MW (trihydrate) = 136.08
pH 5.2
Sodium azide
Sodium azide kdorfman Tue, 12/27/2011 - 18:08For PBS-Tw-Azide
NaN3
MW = 65
10% w/vol
Sodium bicarbonate
Sodium bicarbonate kdorfman Tue, 12/20/2011 - 14:54Sodium bicarbonate (CHNaO3) 0.5 M (=500 mM)
MW = 84.01
Write the date on it. Probably only stable for 2 weeks.
Sodium chloride
Sodium chloride kdorfman Tue, 12/20/2011 - 14:36Sodium Chloride (NaCl) 5 M
MW = 58.44
Sodium ferric EDTA
Sodium ferric EDTA kdorfman Mon, 08/03/2015 - 18:43NaFe(III)EDTA
FW = 367.05
50 mM
Sodium hydroxide
Sodium hydroxide kdorfman Mon, 10/29/2018 - 17:55NaOH
MW = ~40
10mM = 0.01M = 0.4g/L
Sodium phosphate dibasic
Sodium phosphate dibasic kdorfman Tue, 12/20/2011 - 15:00Use 0.2M for colorimetric exercise
Aliquote ~400 uL for a pair of students to each do the exercise
Use hot water
Sodium phosphate monobasic
Sodium phosphate monobasic kdorfman Tue, 12/20/2011 - 14:52Sodium phosphate monobasic (NaH2PO4)
MW (monohydrate) = 137.99
Use 0.2M for colorimetric exercise
Aliquot ~400 uL - enough for 2 students to each do one exercise.
Sodium sulfate
Sodium sulfate kdorfman Thu, 03/01/2012 - 14:17Na2SO4.10H2O
MW = 322.2
Spermidine
Spermidine kdorfman Fri, 05/31/2013 - 18:21Spermidine:
- neutralizes and stabilizes the negative charge on the DNA phosphate backbone
- displaces other ions from DNA
- stimulates restriction enzyme reactions
- increases specificity and reproducibility of Taq-mediated PCR
Used in Gene & Genome
1 g spermidine (entire contents of bottle) in 6.9 mL water
MW = 145.25
Filter sterilize (0.22 µm filter, 10 mL syringe) Freeze 200 uL aliquots
1 M stock
1 g spermidine (entire contents of bottle) in 6.9 mL water
MW = 145.25
Make 20 mM solution for students by adding 800 uL to the 200 uL of 1M in the tube, then aliquot 10 uL for each student.
20 mM student solutions
TBSTw
TBSTw kdorfman Thu, 11/17/2022 - 17:24Tris Buffered Saline with Tween
- Tris 20 mM
- NaCl 100 mM
- Tween 0.1%
- pH 7.4
Tris
Tris kdorfman Tue, 12/20/2011 - 14:42Tris 1M
Tris base MW = 121.14
pH to 7.5 or 8.0, depending on the application
Tris-HCl
Tris-HCl kdorfman Tue, 06/21/2022 - 17:26MW 157.6
Tris-HCl 1M
Triton X
Triton X kdorfman Tue, 12/20/2011 - 15:17Triton X 10%
MW = 646.86
1 g = ~1 mL
Tryptophan
Tryptophan kdorfman Wed, 01/15/2020 - 21:4250X Tryptophan
7.5 g/L
Dissolve 1.875 g Tryptophan in 250 mL of distilled water (reserve some water to rinse the sides of the beaker). Leave it on the stir plate for a while. It's powdery and resistant to going into solution.
Filter sterilize.
Store in the dark at 4 ˚C.
Tween 10%
Tween 10% kdorfman Tue, 12/27/2011 - 18:01Tween 10%
for PBS-Tween-Azide
1 g = ~1 mL [density = 1.095 g/mL at 25 °C (from Sigma website)]
Do not autoclave
Sugars
Sugars kdorfman Mon, 09/12/2016 - 21:45Glucose
Glucose kdorfman Mon, 09/12/2016 - 22:03Molarity (MW = 198.17)
Then bring to final volume. Filter sterilize
Percent
Then bring to final volume. Filter sterilize
Lactose
Lactose kdorfman Mon, 09/12/2016 - 22:07MW = 342.3
heat water first, then tap in lactose while stirring.
Mannitol
Mannitol kdorfman Mon, 09/12/2016 - 22:08MW = 182.17
Sucrose
Sucrose kdorfman Mon, 09/12/2016 - 22:02MW = 342.3
Solubility: 100 mg/mL in water at RT (= 10 g/100 mL)