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

Table of antibiotic stability in poured agar

LB + sugars for Lac-operon work

Filter sterilize

Add 50 µg/mL Kanamycin as indicated here.

MacConkey 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

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

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

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

Make 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.

For motility strains

FBS comes in 500 mL bottles.

Aliquot:

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

Anti-Anti comes in 100 mL bottles.

Aliquot:

Keep frozen until ready to use.

Fisher SV30079.01, $20

from Kathleen Arcaro

bring to final volume, filter sterilize in BSC, refrigerate

insulin solution 10 mg/mL in HEPES

amino acid solution

Medium 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

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.

for all LLCPk cell lines

Thaw FBS and anti-anti first!

bring to final volume, filter sterilize in BSC, refrigerate

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

Medium 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 (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

Try this: http://www.thermofisher.com/us/en/home/life-science/cell-culture/mammal…

FluoroBrite™ DMEM Media

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

1 gram of methylene blue per liter of RO water.

Shake well to dissolve.

Store at room temp.

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

Mark with 2 red lines

Add 1 black line for carbenicillin (ampicillin)

Add 1 green line for IPTG

If required, add IPTG 0.5mM (0.5 mL 1M per L medium) to 1 mM (1 mL 1M per L medium),

and ampicillin

DIW (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.

1/2 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

C Fern Manual

Carolina C-Fern materials

Pre-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

Lilly pollen growth medium

205 mM (7%) sucrose (See Sugars.)

1.6 mM H3BO3

0.1 mM CaCl2

15 mM MES

Either make fresh each day, or filter sterilize for all the labs.

Stock solutions:

150 mM MES

10 mM CaCl2

160 mM Borate

150 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

Murashige & 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

Murashige & 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

Wikipedia entry

Before 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

Per 1 L

• 100 mL 10X M9 salts
• 240 mL glycerol
• 300 µL 1M MgSO4
• water to 1L

Filter sterilize

Per 1 L:

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

Filter sterilize

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.

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

For 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

Minimal 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

Plates for Jeff Laney's Cell & Molecular Biology Lab

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

SC minus (adenine, histidine, leucine, tryptophan)

Sunrise Science 1330-030

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.

Concentrate 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

Liquid 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:

Filter sterilize

Store at room temperature

Yeast 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.

Yeast 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.

Sporulation 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 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

A Dohlman Lab Protocol

Yeast 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.-

1. Grow a starter culture at 30 with shaking (250 rpm) until it reaches saturation.

2. In a 1.8 ml cryotube, mix 0.5 ml of the saturated culture with 0.5 ml of YPD containing 20% glycerol.

3. Flash freeze the tube in liquid nitrogen and store at -80.

4. 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.

5. Allow the yeast to grow at 30 until colonies appear (2-6 days).

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.

Yeast extract-Peptone-Dextrose medium: