Culture Media

Culture Media kdorfman Fri, 04/27/2018 - 15:16

Bacterial media

Bacterial media kdorfman Thu, 01/17/2013 - 18:53

Freezing bacteria

Freezing bacteria kdorfman Sat, 12/05/2015 - 18:44

To 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 agar

LB agar kdorfman Thu, 01/17/2013 - 18:55

LB agar + broth

LB agar + broth kdorfman Tue, 06/25/2019 - 20:00

25 g LB + 15 g agar /liter

To make LB agar plates (100 mm)
you'll need: mL water
add: g LB broth, stir till dissolved
add: g agar, leave stir bar in
autoclave: minutes

LB agar granules

LB agar granules kdorfman Tue, 06/25/2019 - 20:01

Make with granulated LB (Miller) agar, Fisher BP9724

To make LB agar plates
at mm plate diameter
pour mL per plate
start with: mL water
addg LB agar *granules*, stir till dissolved
autoclave for: minutes

LB agar powder

LB agar powder kdorfman Tue, 06/25/2019 - 20:00

Make with LB (Miller) Agar powder, Difco 244510

To make LB agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add g LB agar, stir till dissolved
autoclave for: minutes

LB antibiotic

LB antibiotic kdorfman Wed, 10/01/2014 - 16:34
To make: mL LB
with: µg/mL antibiotic
and the antibiotic stock is: mg/mL
then add: µL antibiotic stock solution (after hot medium can be handled)

LB broth

LB broth kdorfman Tue, 05/28/2013 - 17:48

LB Broth Miller Luria-Bertani

Fisher DF0446-17-3 500 g $40.61.

25 g/L

To make mL LB broth
add: grams LB mix, stir till dissolved.
autoclave: minutes

LB-sugars

LB-sugars kdorfman Wed, 10/01/2014 - 16:19

LB + sugars for Lac-operon work

sugar (or analog) MW
mono saccharide 180.2
disaccharide 342.3
IPTG 238.3

glucose, galactose, or fructose
To make mL LB broth
that is: M sugar,
add: g monosaccharide (glucose, galactose, or fructose)

lactose, maltose, sucrose
To make mL LB broth
that is: M sugar,
add: g disaccharide (lactose, maltose, sucrose)

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.

IPTG
To make mL LB broth
that is: mM IPTG,
add: µL 1M IPTG

Filter sterilize

Add 50 µg/mL Kanamycin as indicated here.

MacConkey agar

MacConkey agar kdorfman Thu, 01/17/2013 - 19:41

MacConkey Agar

  • Fisher 212122 2 kg (Difco)
  • Krackeler 10-211387 500g (via Sigma)

to distinguish Lac+ and Lac- bacterial strains.

50 g/L

To make Maconkey agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add g Maconkey agar, heat to boiling, stir till dissolved
autoclave for: minutes

leave stir bar in

put bottles on stir plate near sterile hood until handle-able

Min med for GFP cells

Min med for GFP cells kdorfman Tue, 10/23/2018 - 15:53

From https://wahoo.cns.umass.edu/node/857

For SfGFP cells, try M9 + 0.2% glycerol + 0.01 mM FeSO4

To make mL min med
start with mL 10X M9
add: mL 100% glycerol
add: mL 10mM FeSO4
Bring to final volume, filter sterilize

Mueller Hinton agar

Mueller Hinton agar kdorfman Thu, 01/17/2013 - 20:05

90922 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

To make Mueller-Hinton plates (100 mm)
you'll need: mL water
add: g MH broth, stir till dissolved
add: g agar, leave stir bar in
autoclave: minutes

P-Glo Bacterial media

P-Glo Bacterial media kdorfman Mon, 08/26/2019 - 22:26

Make 60 mm plates

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 - 22:27

LB 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 - 17:34

For motility strains

To make mL min med
start with mL 10X M9
add: mL 200X HMLTT
add: mL 100% glycerol
add: mL H2O
add: mL 1M MgSO4
add: uL 1M CaCl2, stir if it precipitates
Bring to final volume

Cell culture media

Cell culture media kdorfman Tue, 10/11/2011 - 19:24

FBS aliquots

FBS aliquots kdorfman Tue, 10/11/2011 - 20:15

FBS 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 - 20:21

Anti-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

DMEM

DMEM kdorfman Tue, 10/11/2011 - 19:30

Medium for 3t3 fibroblasts and B16 cells

To make mL DMEM
start with mL water
add: g DMEM*
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: anti/anti

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 - 19:49

for all LLCPk cell lines

Thaw FBS and anti-anti first!

To make mL F10-Ham's
start with mL water
add: g F10 (Hams) (Sigma N6635)
add: g Optimem (Invitrogen 226000-050)
add: g NaHCO3
add: g HEPES, then pH to 7.2 (initial pH = ~7.1)
add: mL FBS
add: mL anti/anti

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 - 20:42

Medium with 15% DMSO and 20% serum to protect cells in liquid nitrogen.

F10 Hams for freezing (initial serum concentration = 0.075)

To make mL F10-Ham's for freezing
add: mL F-10 Ham's
add: mL serum
add: mL DMSO

DMEM for freezing (initial serum concentration = 0.1)

To make mL DMEM for freezing
add: mL DMEM
add: mL serum
add: mL DMSO

Filter sterilize

Non-CO2 Media

Non-CO2 Media kdorfman Tue, 12/27/2011 - 18:37

serum-free

serum-free kdorfman Wed, 10/19/2011 - 15:07

Non-CO2 serum-free medium

For working with live cells at the microscope

To make mL non CO2 medium
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL anti/anti

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 - 20:00

Non-CO2 Medium

For working with live cells at the microscope, when serum is needed for normal division.

To make mL F10-Ham's
start with mL water
add: g MEM (Sigma M3024-1L)
add: g HEPES, then pH to 7.3 (initial pH = ~6.3)
add: mL Na pyruvate 100 mM (thermo SH30239.01)
add: mL FBS
add: mL anti/anti

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 - 18:58

Try 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 - 21:48

Use 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 - 14:53

1 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 - 14:54

E2A (100x)

E2A (100x) kdorfman Thu, 03/19/2020 - 14:51

FOLLOW 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

To make mL E2 medium
add: mL 5 M NaCl
add: mL 1 M KCl
add: mL 1 M MgSO4
add: mL 1 M KH2PO4
add: mL 1M Na2HPO4

E2A Buffer Mix

E2A Buffer Mix kdorfman Thu, 03/19/2020 - 14:52

E2B 500X

E2B 500X kdorfman Thu, 03/19/2020 - 14:52

E2C 500X

E2C 500X kdorfman Thu, 03/19/2020 - 14:53

VTM

VTM kdorfman Fri, 04/03/2020 - 14:42

Viral 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

Worm media

Worm media kdorfman Fri, 04/01/2016 - 16:28

Freezing medium

Freezing medium kdorfman Fri, 04/01/2016 - 16:30

Per 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 - 18:11

Per 1 L:

  • 100 mL 10X M salts
  • 300 µL 1M MgSO4
  • water to 1 L

Filter sterilize

NGM

NGM kdorfman Fri, 04/01/2016 - 16:31

Per 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.

To make NGM agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g peptone, stir till dissolved
addg NaCL, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add: mL 5mg/mL cholesterol
add: mL sterile 1M CaCl2
add: mL sterile 5mg/mL 1M MgSO4
add: mL sterile K-phosphate buffer , pH 6

put bottles on stir plate near sterile hood until handle-able


  1. 3.3 mL K2HPO4 + 21.7 mL KH2PO4 ↩︎

Yeast media

Yeast media kdorfman Mon, 02/08/2016 - 15:03

MV + Ade

MV + Ade kdorfman Wed, 10/18/2017 - 16:42
  • 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.

To make MV+ADE agar plates
pour mm plate diameter
pour mL per plate
start with: mL water
add: g YNB*, stir till dissolved
add: g ammonium sulfate, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
add asceptically: mL filter-sterilized Adenine (1mg/mL)
for a final volume of mL

Mark with two blue lines

*Yeast Nitrogen Base without amino acids and ammonium sulfate

MV

MV kdorfman Mon, 02/08/2016 - 15:04

Minimal Vitamin Medium

Autoclave an appropriately sized graduated cylinder to measure the glucose solution.

To make MV agar plates
pour mm plate diameter
pour mL per plate
start with: mL water
add: g Yeast Nitrogen Base without amino acids and ammonium sulfate, stir till dissolved
add: g ammonium sulfate, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

Mark with a single blue line

SC -Leu High Ade

SC -Leu High Ade kdorfman Sun, 01/19/2020 - 16:39

Plates for Jeff Laney's Cell & Molecular Biology Lab

Laney's SC high ade

Laney's SC high ade kdorfman Sun, 01/26/2020 - 16:22

From 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 - 16:26

SC -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 - 16:23

Using 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.

To make
SC-leu high ade agar plates
with mm plate diameter
pour mL per plate
start with: mL water
add: g agar, leave stir bar in
autoclave for: minutes
Meanwhile, using X SC-AHLT
Asceptically mix together,
warm up, then add to autoclaved agar:
mL SC-AHLT,
and : mL 10x YNB+AS ,
and: mL 20% glucose ,
and: mL 10X Adenine hemi sulfate,
and: mL 50X Tryptophan,
and: mL 100X Histidine HCl.
for a final volume of mL

Mark with one green line

SC-AHLT high Ade mostly powder

SC-AHLT high Ade mostly powder kdorfman Tue, 02/04/2020 - 13:41

High 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 - 22:07

SC-Leu low-Ade plates for Jeff Laney's cell and molecular biology lab

Laney's original

Laney's original kdorfman Sun, 01/26/2020 - 17:02

SC-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 - 17:02

SC -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 - 17:01

Using 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.)

To make SC-leu low ade agar plates
with mm plate diameter
pour mL per plate
start with: mL water
add: g agar, leave stir bar in
autoclave for: minutes
Meanwhile, using X SC-AHLT
Asceptically mix together,
warm up, then add to autoclaved agar:
mL SC-AHLT,
and : mL 10X YNB+AS,
and: mL 20% Glucose,
and: mL 10x adenine hemi sulfate,
and: mL 50X Tryptophan
and: mL 100X Histidine .
for a final volume of mL

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 - 13:26

High 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

SC-AHLT

SC-AHLT kdorfman Wed, 01/15/2020 - 21:45

SC minus (adenine, histidine, leucine, tryptophan)

Sunrise Science 1330-030

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.


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


4X will dissolve. Almost completely clear after overnight stirring. Crystal clear after autoclaving.

SC-complete-high-Ade

SC-complete-high-Ade kdorfman Wed, 01/15/2020 - 22:12

Liquid Medium

Jeff Laney's 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


Using a different concentration of SC-AHLT (because 10x won't go into solution):

To make mL SC-complete high ade liquid medium
Meanwhile, using X SC-AHLT
Mix: mL 10X SC–AHLT,
and : mL 10X YNB+AS,
and: mL 20% Glucose,
and: mL 10x adenine hemi sulfate,
and: mL 50X Tryptophan ,
and: mL 100X Histidine .
and: mL 10X Leucine

Bring to final volume with water, then filter sterilize.

SC -Leu High Ade

SC -Leu High Ade kdorfman Sun, 01/19/2020 - 13:33

Recipe 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:

To make
SC-leu high ade agar plates
with mm plate diameter
pour mL per plate
start with: mL water
add: g agar, leave stir bar in
autoclave for: minutes
Meanwhile, using X SC-AHLT
Asceptically mix together,
warm up, then add to autoclaved agar:
mL SC-AHLT,
and : mL 10x YNB+AS ,
and: mL 20% glucose ,
and: mL 10X Adenine hemi sulfate,
and: mL 50X Tryptophan,
and: mL 100X Histidine HCl.
for a final volume of mL

Mark with one green line

YED

YED kdorfman Mon, 02/08/2016 - 19:22

Yeast Extract Dextrose

Remember to autoclave an appropriate sized graduated cylinder to measure the glucose solution in the sterile hood.

To make YED agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL

Mark with a single red line.

YEKAC

YEKAC kdorfman Mon, 02/08/2016 - 19:29

Sporulation Medium

To make YEKAC agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g potassium acetate, stir till dissolved
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes

Mark with a single black line.

YNB + AS

YNB + AS kdorfman Wed, 01/15/2020 - 21:44

10X 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 - 15:04

Yeast extract-Peptone-Dextrose medium:

To make YPD agar plates
at mm plate diameter
pour mL per plate
start with: mL water
add: g peptone broth, stir till dissolved
add: g yeast extract, stir till dissolved
add: g agar, leave stir bar in
autoclave for: minutes
add asceptically mL filter sterilized 20% glucose
for a final volume of mL YPD agar