Team:Caltech/Recipes

From 2011.igem.org

(Difference between revisions)
Line 42: Line 42:
<p>'''Gibson Mix (1.33x)'''</p>  
<p>'''Gibson Mix (1.33x)'''</p>  
 +
For 25 aliquots of 15 μl each:
* 50 μl of Taq Ligase  
* 50 μl of Taq Ligase  
* 100 μl of 5x isothermal buffer
* 100 μl of 5x isothermal buffer
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* 216.75 μl of Nuclease-free water  
* 216.75 μl of Nuclease-free water  
* (375 μl total)  
* (375 μl total)  
-
 
+
<br/>
-
This makes 25 aliquots of 15 μl each.
+
-
<br/><br/>
+
<p>'''Phusion PCR'''</p>
<p>'''Phusion PCR'''</p>
 +
For a 50uL reaction:
* 1 uL template DNA
* 1 uL template DNA
* 2..5 uL fwd and rev primer
* 2..5 uL fwd and rev primer
Line 68: Line 68:
*adjust pH to 7.5 using 1M NaOH
*adjust pH to 7.5 using 1M NaOH
*autoclave
*autoclave
-
*after cooling below 50 degrees Celsius, add 5 mL filter-sterilized 20% glucose solution.
+
*after cooling below 50&deg;C, add 5 mL filter-sterilized 20% glucose solution.<br/><br/>
 +
 
 +
<p>'''Taq PCR (for 16s insert)'''</p>
 +
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
<br/>}}
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Revision as of 21:59, 27 July 2011


Caltech iGEM 2011



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50% glycerol Stock:

  • 50 mL glycerol
  • 50 mL nanopure water


Agar/LB plate (Autoclaved):

  • 500 mL 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

  • 1.0712 g K2 HPO4
  • 0.5239 g KH2 PO4
  • 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


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)


Phusion PCR

For a 50uL reaction:

  • 1 uL template DNA
  • 2..5 uL fwd and rev primer
  • 19 uL sterile water
  • 25 uL Phusion master mix

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.

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



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