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Team:Stanford/Protocols
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Digestion Recipe
| DNA: | 2 ug | (calculate volume based off concentration obtained from nanodrop) |
| 10x BSA: | 5 uL | (if needed, check NEB website) |
| 10x NEBuffer: | 5 uL | (use [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp the NEB website] to find which buffer) |
| Each enzyme: | 1 uL | (add last; it's the most expensive ingredient) |
| Sterile water: | ~26 uL | (so that total volume is 50uL) |
Ligation Recipe
| Vector DNA: | 150 ng | |
| 5 - 20x insert DNA: | __ ng | --> (5 to 20) * (insert bp / vector bp) * 150 see [http://bioinfo.clontech.com/infusion/molarRatio.do this calculator] for help |
| 10x T4 ligase buffer: | 2 uL | |
| T4 ligase: | 1 uL | (add last; it's the most expensive ingredient) |
| Sterile water: | __ uL | (so that total volume is 20uL) |
PCR Assembly
| Forward piece | 5 uL |
| Backward piece | 5 uL |
| PCR Supermix | 40 uL |
Electroporation Transformation
Cells: E. coli DH10B
Protocol:
- Get DNA (0.5 to 2.0 uL)
- Transfer DNA to electrocompetent cells (keep cells in ice bucket)
- Resuspend cells and DNA
- Transfer cells and DNA to cuvette (don't leave air bubbles, and hold cuvette at the top to keep the bottom cool)
- Zap the cells (2000V, 25uF, 200ohms)
- Add 1000uL SOC to cuvette, resuspend the cells, and transfer to microfuge tube
- Incubate cells at 37oC for 30 to 60 minutes. Warm up agar plates in anticipation.
- Centrifuge the cells, remove 800uL of supernatant, resuspend cells in remaining 200uL.
- Plate the cells, incubate at 37oC for 12-16 hours.
Heat-Shock Transformation
Cells: E. coli DH5alpha
Protocol:
- Take out competent E.coli cells from –80 C freezer.
- Turn on water bath to 42 C.
- Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar) – you may need more or less cells, depending how competent they are.
- Keep tubes on ice.
- Add 50 ng or 2.5 ul of circular DNA into E.coli cells. Incubate on ice for 30 min to thaw competent cells.
- Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
- Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
- Add 1 ml of SOC. Incubate tubes for 2 hours at 37 C.
- Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight.
- Pick the colonies about 12-16 hours later.
