Team:Caltech/Recipes

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)

• 50 μl of Taq Ligase
• 100 μl of 5x isothermal buffer
• 2 μl of T5 exconuclease
• 5 μl of Phusion polymerase
• Nuclease-free water
• 15 μl of Master Mix Total

Phusion PCR

• 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 degrees Celsius, add 5 mL filter-sterilized 20% glucose solution.