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From 2010.igem.org

Media Recipes

LB media

• 10 g LB broth
• 500 mL DI water

autoclave to sterilize

LB media with agar

• 10 g LB broth
• 7.5 g agar
• 500 mL DI water

autoclave to sterilize

add antibiotics after autoclaving when warm to touch (about 50°C)

pour plates when warm to the touch (about 50°C)

0.1 M CaCl2 solution

• 0.735 g of CaCl2*2 H20
• 50 mL of DI water

filter sterilize

1.0 M CaCl2 solution

• 7.35 g of CaCl2*2 H20
• 50 mL of DI water

filter sterilize

1.0 M MgSO4

• 12.323 g of MgSO4*7 H20
• 50 mL of DI water

filter sterilize

Ampicillin Antibiotic Stock Solution (100 mg/mL)

• 1 g of ampicillin
• 5 mL of DI water
• 5 mL of ethanol

filter sterilize and make 1 mL aliquotes to store in the -20°C freezer

Kanamycin Antibiotic Stock Solution (50 mg/mL)

• 250 mg of kanamycin
• 5 mL of DI water

filter sterilize and make 1 mL aliquots to store in the -20°C freezer

Tetracycline Antibiotic Stock Solution (50 mg/mL)

• 250 mg of tetracycline
• 2.5 mL of DI water
• 2.5 mL of ethanol

filter sterilize and make 1 mL aliquots to store in the -20°C freezer

IPTG stock solution (1 M)

• 0.25 g of IPTG
• 1049 uL of DI water

filter sterilize with .22 um filter

1.5 mg/mL Cyclohexanecarboxylic acid solution

Cyclohexanecarboxylic acid is volatile should only be worked with in a hood. For accurate measurement use covered scale with sliding doors in the hood. Stored in the corrosive cabinet

• 0.075 g cyclohexanecarboxylic acid
• 50 mL of DI water

filter sterilize

Bushnell-Haas Salts (w/out carbon source)

• 0.4 g of KH2PO4
• 0.4 g of K2HPO4
• 0.4 g of (NH4)2 SO4 or NH4NO3
• 0.002 g of FeCl3
• 318 mL of DI H2O

Autoclave at 15 psi and 121C for 30 minutes

After the media has cooled completely add:

• 665 uL of 1 M MgSO4 solution
• 72 uL of 1 M CaCl2 solution

40% glucose solution

• 20 g of glucose
• 50 mL of DI water

filter sterilize

Bushnell-Haas Media with glucose

• 39.75 mL of Bushnell-Haas Salts
• 2 mL of 40% glucose solution
• 8.25 mL of sterile DI water

Bushnell-Haas Media with cyclohexanecarboxylic acid

• 39.75 mL of Bushnell-Haas Salts
• 4 mL of cyclohexanecarboxylic acid solution
• 6.25 mL of sterile DI water

20 mg/mL Casamino Acid Solution

• 0.18 g Casamino acid
• 9 mL of DI water

Filter sterilize

Bushnell-Haas Media with glucose and casamino acids

• 39.75 mL of Bushnell-Haas Salts
• 2 mL of 40% glucose solution
• 2 mL of sterile DI water
• 6.25 mL of casamino acid solution

0.1 M NaOH solution

BE VERY CAREFUL WHEN MIXING! SOLID NaOH BURNS YOUR SKIN BUT YOU MAY NOT FEEL IT AT FIRST! MAKE SURE ANY SPILLS ARE CLEANED UP VERY WELL!

• 0.2 g of NaOH
• 50 mL of sterile water

Filter Sterilize

Bushnell-Haas Media with cyclohexanecarboxylic acid adjusted to pH of 9

• 39.75 mL of Bushnell-Haas Salts
• 4 mL of cyclohexanecarboxylic acid solution
• 6.25 mL of sterile DI water

Test how much 0.1 M NaOH must be added to 1 mL of media to raise it to a pH of 9 (add 1 uL of base at a time)

Scale up the amount of 0.1 M NaOH solution to add to the remaining volume of media (approaching final amount slowly)

On 8/3/2010 approximately 20 uL of 0.1 M NaOH had to be added to 1 mL to get to a pH between 8 and 9 (tested with pH paper) and 675 uL of 0.1 M NaOH was added into the 45 mL of remaining media.