Team:Caltech/Recipes

From 2011.igem.org

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==Chloramphenicol Stock==
==Chloramphenicol Stock==
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*for 25 mg/ml stock solution (usually, chloramphenicol is used at 12.5 µg/ml or 25 µg/ml)
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*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
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*add 250 mg chloramphenicol to 10 ml 100% ethanol
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*Store at -20°C
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*store at -20°C
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Revision as of 23:04, 28 September 2011


Caltech iGEM 2011



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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

  • 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)

IPTG stock

For 1000x stock (.1M)

  • .0238 g IPTG
  • 1 mL sterile water

Ligation Reaction

for 20 uL rxn

  • 2 uL T4 buffer
  • x mols insert
  • 3x mols vector
  • 1 uL T4 ligase

H&subO make up 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 fwd and rev 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 50uL rxn

  • 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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