Team:Michigan/Media Recipes

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Latest revision as of 23:52, 26 October 2010

Media Recipes

General Recipes

Oil Sands Recipes

General Recipes

LB media

10 g LB medium
500 mL DI water

autoclave to sterilize

LB media with agar

10 g LB medium
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

40% glucose solution

20 g of glucose
50 mL of DI water

filter sterilize

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

10mM dNTP mix for PCR

MAKE SURE ALL REAGENTS ARE WELL MIXED BEFORE STARTING BY PULSE CENTRIFUGING OR FLICKING

100 uL of 100mM dATP
100 uL of 100mM dTTP
100 uL of 100mM dGTP
100 uL of 100mM dCTP
600 uL of ultra pure water

aliquot into 50 uL volumes (this will take 20 1.5mL eppendorf tubes)

Oil Sands Recipes

1.5 mg/mL Cyclohexanecarboxylic acid solution

Cyclohexanecarboxylic acid is volatile and should only be used under a hood. For accurate measurement, use a covered scale with sliding doors (in the hood). Store 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 121°C for 30 minutes

After the media has cooled completely add:

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

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

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.

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+{|cellspacing=0
+|-valign="top"
+|width="700px" style="background-color:rgb(190,210,250);"|
 ==Media Recipes==
+[[Team:Michigan/Media Recipes#General Recipes|General Recipes]]
+[[Team:Michigan/Media Recipes#Oil Sands Recipes|Oil Sands Recipes]]
+==General Recipes==
 '''LB media'''
-*10 g LB broth
+*10 g LB medium
 *500 mL DI water
 ''autoclave to sterilize''
 '''LB media with agar'''
-*10 g LB broth
+*10 g LB medium
 *7.5 g agar
 *500 mL DI water
 ''autoclave to sterilize''
-''add antibiotics when warm to touch (about 50C)''
+''add antibiotics after autoclaving when warm to touch (about 50°C)''
-''pour when warm to the touch (about 50C)''
+''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 -20C freezer''
+''filter sterilize and make 1 mL aliquots to store in the -20°C freezer''
 '''Tetracycline Antibiotic Stock Solution (50 mg/mL)'''
@@ Line 34: / Line 66: @@
 *2.5 mL of DI water
 *2.5 mL of ethanol
-''filter sterilize and make 1 mL aliquots to store in the -20C freezer''
+''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''
+'''40% glucose solution'''
+*20 g of glucose
+*50 mL of DI water
+''filter sterilize''
+'''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''
+'''10mM dNTP mix for PCR'''
+''MAKE SURE ALL REAGENTS ARE WELL MIXED BEFORE STARTING BY PULSE CENTRIFUGING OR FLICKING''
+*100 uL of 100mM dATP
+*100 uL of 100mM dTTP
+*100 uL of 100mM dGTP
+*100 uL of 100mM dCTP
+*600 uL of ultra pure water
+''aliquot into 50 uL volumes (this will take 20 1.5mL eppendorf tubes)''
+==Oil Sands Recipes==
+'''1.5 mg/mL Cyclohexanecarboxylic acid solution'''
+Cyclohexanecarboxylic acid is volatile and should only be used under a hood.  For accurate measurement, use a covered scale with sliding doors (in the hood).  Store 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 121°C for 30 minutes''
+After the media has cooled completely add:
+*665 uL of 1 M MgSO4 solution
+*72 uL of 1 M CaCl2 solution
+'''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
+'''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.
+|width="250px" style="background-color:rgb(190,210,250)"|
+[[Image:Lab01.png|middle|250px]]
+[[Image:Lab02.png|middle|250px]]
+[[Image:Lab04.png|middle|250px]]
+[[Image:Lab05.png|middle|250px]]
+[[Image:Lab06.png|middle|250px]]
+[[Image:Lab09.png|middle|250px]]
+[[Image:Lab07.png|middle|250px]]
+[[Image:Lab08.png|middle|250px]]
+[[Image:Lab10.png|middle|250px]]
+[[Image:Lab11.png|middle|250px]]
+|}