Team:Michigan/Media Recipes

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Revision as of 02:52, 4 August 2010

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.

@@ Line 119: / Line 119: @@
 *4 mL of cyclohexanecarboxylic acid solution
 *6.25 mL of sterile DI water
-Add  0.1 M NaOH solution 1 uL at a time and dot 1 uL of solution on pH paper until pH reaches 9
+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.