Team:Caltech/Recipes

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
• 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.

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