Team:Michigan/Media Recipes

From 2010.igem.org

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==Media Recipes==
==Media Recipes==
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[[Team:Michigan/Media Recipes#General Recipes|General Recipes]]
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[[Team:Michigan/Media Recipes#Oil Sands Recipes|Oil Sands Recipes]]
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==General Recipes==
'''LB media'''
'''LB media'''
-
*10 g LB broth
+
*10 g LB medium
*500 mL DI water
*500 mL DI water
''autoclave to sterilize''
''autoclave to sterilize''
'''LB media with agar'''
'''LB media with agar'''
-
*10 g LB broth
+
*10 g LB medium
*7.5 g agar
*7.5 g agar
*500 mL DI water
*500 mL DI water
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''filter sterilize with .22 um filter''
''filter sterilize with .22 um filter''
-
'''Cyclohexanecarboxylic acid'''
+
'''40% glucose solution'''
 +
*20 g of glucose
 +
*50 mL of DI water
 +
''filter sterilize''
 +
 
 +
'''0.1 M NaOH solution '''
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 +
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'''
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 +
''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)''
 +
 
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==Oil Sands Recipes==
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'''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
+
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
*0.075 g cyclohexanecarboxylic acid
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*0.002 g of FeCl3
*0.002 g of FeCl3
*318 mL of DI H2O  
*318 mL of DI H2O  
-
Autoclave at 15 psi and 121C for 30 minutes
+
''Autoclave at 15 psi and 121°C for 30 minutes''
After the media has cooled completely add:
After the media has cooled completely add:
*665 uL of 1 M MgSO4 solution
*665 uL of 1 M MgSO4 solution
*72 uL of 1 M CaCl2 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'''
'''Bushnell-Haas Media with glucose'''
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*4 mL of cyclohexanecarboxylic acid solution
*4 mL of cyclohexanecarboxylic acid solution
*6.25 mL of sterile DI water
*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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|width="250px" style="background-color:rgb(190,210,250)"|
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[[Image:Lab01.png|middle|250px]]
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[[Image:Lab02.png|middle|250px]]
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[[Image:Lab04.png|middle|250px]]
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[[Image:Lab05.png|middle|250px]]
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[[Image:Lab06.png|middle|250px]]
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[[Image:Lab09.png|middle|250px]]
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[[Image:Lab07.png|middle|250px]]
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[[Image:Lab08.png|middle|250px]]
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[[Image:Lab10.png|middle|250px]]
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[[Image:Lab11.png|middle|250px]]
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|}

Latest revision as of 23:52, 26 October 2010


Michigan Header





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.

Lab01.png

Lab02.png

Lab04.png

Lab05.png

Lab06.png

Lab09.png

Lab07.png

Lab08.png

Lab10.png

Lab11.png

Retrieved from "http://2010.igem.org/Team:Michigan/Media_Recipes"