Team:Caltech/Recipes
From 2011.igem.org
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- | * | + | [[Team:Caltech/Notebook|Back to Timeline]] . [[Team:Caltech/Protocols|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 tryptone | ||
* 10 g NaCl | * 10 g NaCl | ||
* 5 g yeast extract | * 5 g yeast extract | ||
* 15 g agar | * 15 g agar | ||
- | + | ||
- | + | ==Ampicillin Stock== | |
- | * 50 | + | *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 | * 79 mL Phosphate solution | ||
- | * | + | * 10 mL Salt Solution I |
- | * | + | * 10 mL Salt Solution II |
- | * | + | * 1 mL Wolfe's Vitamin Solution |
- | * 1 mL SL-10 Trace Element Solution. | + | * 0.1 mL SL-10 Trace Element Solution. |
* variable amounts of EDC's | * variable amounts of EDC's | ||
- | |||
- | < | + | Phosphate solution: 1.0712g K2 HPO4 and 0.5239g KH2 PO4 per liter of water<br/> |
- | *for 250 ml (adapted from [http://openwetware.org/wiki/SOC OpenWetWare]) | + | Salt Solution I: 3g NH4Cl, 10g NaCl, 5g KCl, 1.5g Na2SO4 per liter of water<br/> |
+ | Salt Solution II: 4g MgCl2.6H20 and 0.5g CaCl2.2H20 per liter of water<br/> | ||
+ | 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. <br/> | ||
+ | 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<br/> | ||
+ | |||
+ | |||
+ | ==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<sup>+</sup> | ||
+ | *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 | *1.25 g yeast extract | ||
*5 g tryptone | *5 g tryptone | ||
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*adjust pH to 7.5 using 1M NaOH | *adjust pH to 7.5 using 1M NaOH | ||
*autoclave | *autoclave | ||
- | *after cooling below 50 | + | *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: | |
- | * | + | *10mg X-gal |
- | * | + | *0.5mL DMSO}} |
Latest revision as of 00:00, 29 September 2011
Project |
Back to Timeline . Back to Methods
50% glycerol Stock:
Agar/LB plate (Autoclaved):
Ampicillin Stock
Chloramphenicol Stock
Enrichment Minimal MediaFor 100 mL media:
Phosphate solution: 1.0712g K2 HPO4 and 0.5239g KH2 PO4 per liter of water
Gibson Mix (1.33x)For 25 aliquots of 15 μl each:
IPTG stockFor 1000x stock (.1M)
Ligation ReactionFor a 20 uL reaction:
p450 degradation testing solutionFor a 200uL reaction:
Phusion PCRFor a 50uL reaction:
SOC Mediafor 250 ml (adapted from [http://openwetware.org/wiki/SOC OpenWetWare]):
Restriction DigestFor a 50uL reaction:
Taq PCR (for 16s insert)For a 25uL reaction:
X-gal stock (50x)For 20mg/mL, total 0.5mL volume:
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