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: