Team:Caltech/Recipes

From 2011.igem.org

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{{Team:Caltech/templateheader|
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<p>'''Agar/LB plate:'''
 
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* 500 g of nanopure water
 
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* 10 g of Tryptone
 
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* 10 g of NaCl
 
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* 5 g of yeast extract
 
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* 15 g of Agar
 
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2) Shake and then autoclave.</p>
 
-
<p>'''50% Glycerol Stock:'''<br/>
+
[[Team:Caltech/Notebook|Back to Timeline]] . [[Team:Caltech/Protocols|Back to Methods]]
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1) Pour 50 mL of Glycerol and 50 mL of pure water into a graduated cylinder.<br/>
+
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2) Cover with parafilm and turn over a couple of times to mix.<br/>
+
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3) Use the Nalgene vacuum to sterilize the stock.<br/>
+
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4) Use the bunsen burner to keep air sterile and close the cap.</p>
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<p>'''Enrichment Minimal Media'''</p>  
+
==50% glycerol Stock:==
-
* 1.0712 g of K2 HPO4
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* 50 mL glycerol
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* 0.5239 g of KH2 PO4
+
* 50 mL nanopure water
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* 79 mL of Phosphate solution  
+
   
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* 100 mL of Salt Solution I  
+
 
-
* 100 mL of Salt Solution II  
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==Agar/LB plate (Autoclaved):==
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* 10 mL of Wolfe's Vitamin Solution  
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* 1 L nanopure water
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* 1 mL of SL-10 Trace Element Solution.
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* 10 g tryptone
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and then add 5 mg of whatever chemical used in two flasks, one with vitamins and one without.  
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* 10 g NaCl
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}}
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* 5 g yeast extract
 +
* 15 g agar
 +
 +
 
 +
==Ampicillin Stock==
 +
*Mass out amount needed for 50 mg/ml or 100 mg/ml stock of ampicillin trihydrate
 +
*Add a small amount of nanopure water
 +
*Add 1M NaOH until solution turns clear (we are making ampicillin sodium salt in this step, which is much more soluble in water)
 +
*Fill to total volume with nanopure water
 +
 +
 
 +
==Chloramphenicol Stock==
 +
*For 25 mg/ml stock solution (usually, chloramphenicol is used at 12.5 µg/ml or 25 µg/ml, add 250 mg chloramphenicol to 10 ml 100% ethanol
 +
*Store at -20°C
 +
 
 +
 
 +
==Enrichment Minimal Media== 
 +
For 100 mL media:
 +
* 79 mL Phosphate solution  
 +
* 10 mL Salt Solution I  
 +
* 10 mL Salt Solution II  
 +
* 1 mL Wolfe's Vitamin Solution  
 +
* 0.1 mL SL-10 Trace Element Solution.
 +
* variable amounts of EDC's
 +
 
 +
Phosphate solution: 1.0712g K2 HPO4 and 0.5239g KH2 PO4 per liter of water<br/>
 +
Salt Solution I: 3g NH4Cl, 10g NaCl, 5g KCl, 1.5g Na2SO4 per liter of water<br/>
 +
Salt Solution II: 4g MgCl2.6H20 and 0.5g CaCl2.2H20 per liter of water<br/>
 +
Wolfe's Vitamin Solution (100x): 10 mg Pyridoxine.Hcl, 5 mg p-Aminobenzoic acid, 5 mg Lipoic acid, 5 mg Nicotinic acid, 5 mg Riboflavin, 5 mg Thiamine.HCl, 5 mg Pantothenic acid, 2 mg Biotin, 2 mg Folic acid, 0.1 mg Vitamin B12 per liter of water. <br/>
 +
Trace Element Solution:  1500 mg FeCl2.4H2O, 70 mg ZnCl2, 100 mg MnCl2.4H2O, 6 mg H3BO3, 190 mg CoCl2.6H2O, 2 mg CuCl2.2H2O, 24 mg NiCl2.6H2O, 31 mg Na2MoO4 per liter of water<br/>
 +
 
 +
 
 +
==Gibson Mix (1.33x)==
 +
For 25 aliquots of 15 μl each:
 +
* 50 μl of Taq Ligase
 +
* 100 μl of 5x isothermal buffer
 +
* 2 μl of T5 exconuclease
 +
* 6.25 μl of Phusion polymerase
 +
* 216.75 μl of Nuclease-free water
 +
* (375 μl total)
 +
 
 +
==IPTG stock==
 +
For 1000x stock (.1M)
 +
*.0238 g IPTG
 +
*1 mL sterile water
 +
 
 +
==Ligation Reaction==
 +
For a 20 uL reaction:
 +
*2 uL T4 buffer
 +
*x mols insert
 +
*3x mols vector
 +
*1 uL T4 ligase
 +
*Nanopure water to bring volume to 20 uL
 +
*Total of between 50 and 100ng DNA
 +
 
 +
==p450 degradation testing solution==
 +
For a 200uL reaction:
 +
*2mM substrate
 +
*5mM p450
 +
*0.25M glucose
 +
*5mM NADP<sup>+</sup>
 +
*2.30u/mL GDH (glucose dehydrogenase)
 +
*buffer
 +
 
 +
==Phusion PCR==
 +
For a 50uL reaction:
 +
* 1 uL template DNA
 +
* 2..5 uL forward and reverse primer
 +
* 18.5 uL sterile water
 +
* 25 uL Phusion master mix
 +
* .5 uL phusion enzyme
 +
 
 +
==SOC Media==
 +
for 250 ml (adapted from [http://openwetware.org/wiki/SOC OpenWetWare]):
 +
*1.25 g yeast extract
 +
*5 g tryptone
 +
*0.146 g NaCl
 +
*0.0465 g KCl
 +
*0.616 g MgSO4•7H2O
 +
*0.508 g MgCl2•6H2O
 +
*adjust pH to 7.5 using 1M NaOH
 +
*autoclave
 +
*after cooling below 50&deg;C, add 5 mL filter-sterilized 20% glucose solution.
 +
 
 +
==Restriction Digest==
 +
For a 50uL reaction:
 +
*as much DNA as needed
 +
*0.5uL buffer
 +
*5 µl BSA
 +
*1 uL restriction enzyme each
 +
 
 +
==Taq PCR (for 16s insert)==
 +
For a 25uL reaction:
 +
*sterile water: 19.8uL
 +
*taq buffer (10x): 2.5uL
 +
*dNTP (10mM): 0.6uL
 +
*Fwd primer (10uM): 0.5uL
 +
*Rev primer (10uM): 0.5uL
 +
*template DNA : 1uL
 +
*Taq (5U/ul): 0.1uL
 +
 
 +
==X-gal stock (50x)==
 +
For 20mg/mL, total 0.5mL volume:
 +
*10mg X-gal
 +
*0.5mL DMSO}}

Latest revision as of 00:00, 29 September 2011


Caltech iGEM 2011



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Back to Timeline . Back to Methods

Contents

50% glycerol Stock:

  • 50 mL glycerol
  • 50 mL nanopure water


Agar/LB plate (Autoclaved):

  • 1 L nanopure water
  • 10 g tryptone
  • 10 g NaCl
  • 5 g yeast extract
  • 15 g agar


Ampicillin Stock

  • Mass out amount needed for 50 mg/ml or 100 mg/ml stock of ampicillin trihydrate
  • Add a small amount of nanopure water
  • Add 1M NaOH until solution turns clear (we are making ampicillin sodium salt in this step, which is much more soluble in water)
  • Fill to total volume with nanopure water


Chloramphenicol Stock

  • For 25 mg/ml stock solution (usually, chloramphenicol is used at 12.5 µg/ml or 25 µg/ml, add 250 mg chloramphenicol to 10 ml 100% ethanol
  • Store at -20°C


Enrichment Minimal Media

For 100 mL media:

  • 79 mL Phosphate solution
  • 10 mL Salt Solution I
  • 10 mL Salt Solution II
  • 1 mL Wolfe's Vitamin Solution
  • 0.1 mL SL-10 Trace Element Solution.
  • variable amounts of EDC's

Phosphate solution: 1.0712g K2 HPO4 and 0.5239g KH2 PO4 per liter of water
Salt Solution I: 3g NH4Cl, 10g NaCl, 5g KCl, 1.5g Na2SO4 per liter of water
Salt Solution II: 4g MgCl2.6H20 and 0.5g CaCl2.2H20 per liter of water
Wolfe's Vitamin Solution (100x): 10 mg Pyridoxine.Hcl, 5 mg p-Aminobenzoic acid, 5 mg Lipoic acid, 5 mg Nicotinic acid, 5 mg Riboflavin, 5 mg Thiamine.HCl, 5 mg Pantothenic acid, 2 mg Biotin, 2 mg Folic acid, 0.1 mg Vitamin B12 per liter of water.
Trace Element Solution: 1500 mg FeCl2.4H2O, 70 mg ZnCl2, 100 mg MnCl2.4H2O, 6 mg H3BO3, 190 mg CoCl2.6H2O, 2 mg CuCl2.2H2O, 24 mg NiCl2.6H2O, 31 mg Na2MoO4 per liter of water


Gibson Mix (1.33x)

For 25 aliquots of 15 μl each:

  • 50 μl of Taq Ligase
  • 100 μl of 5x isothermal buffer
  • 2 μl of T5 exconuclease
  • 6.25 μl of Phusion polymerase
  • 216.75 μl of Nuclease-free water
  • (375 μl total)

IPTG stock

For 1000x stock (.1M)

  • .0238 g IPTG
  • 1 mL sterile water

Ligation Reaction

For a 20 uL reaction:

  • 2 uL T4 buffer
  • x mols insert
  • 3x mols vector
  • 1 uL T4 ligase
  • Nanopure water to bring volume to 20 uL
  • Total of between 50 and 100ng DNA

p450 degradation testing solution

For a 200uL reaction:

  • 2mM substrate
  • 5mM p450
  • 0.25M glucose
  • 5mM NADP+
  • 2.30u/mL GDH (glucose dehydrogenase)
  • buffer

Phusion PCR

For a 50uL reaction:

  • 1 uL template DNA
  • 2..5 uL forward and reverse primer
  • 18.5 uL sterile water
  • 25 uL Phusion master mix
  • .5 uL phusion enzyme

SOC Media

for 250 ml (adapted from [http://openwetware.org/wiki/SOC OpenWetWare]):

  • 1.25 g yeast extract
  • 5 g tryptone
  • 0.146 g NaCl
  • 0.0465 g KCl
  • 0.616 g MgSO4•7H2O
  • 0.508 g MgCl2•6H2O
  • adjust pH to 7.5 using 1M NaOH
  • autoclave
  • after cooling below 50°C, add 5 mL filter-sterilized 20% glucose solution.

Restriction Digest

For a 50uL reaction:

  • as much DNA as needed
  • 0.5uL buffer
  • 5 µl BSA
  • 1 uL restriction enzyme each

Taq PCR (for 16s insert)

For a 25uL reaction:

  • sterile water: 19.8uL
  • taq buffer (10x): 2.5uL
  • dNTP (10mM): 0.6uL
  • Fwd primer (10uM): 0.5uL
  • Rev primer (10uM): 0.5uL
  • template DNA : 1uL
  • Taq (5U/ul): 0.1uL

X-gal stock (50x)

For 20mg/mL, total 0.5mL volume:

  • 10mg X-gal
  • 0.5mL DMSO


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