Back to Timeline . Back to Methods
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:
"
