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Team:Stanford/Protocols
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==Digestion Recipe== | ==Digestion Recipe== | ||
{| | {| | ||
| - | |DNA: || 12uL | + | |DNA: || align="left" | 12uL |
|- | |- | ||
| - | |10x BSA: || 5uL || (if needed, check NEB website) | + | |10x BSA: || align="left" | 5uL || (if needed, check NEB website) |
|- | |- | ||
| - | |10x NEBuffer 2: || 5uL || (works with E, X, S, P) | + | |10x NEBuffer 2: || align="left" | 5uL || (works with E, X, S, P) |
|- | |- | ||
| - | |Each enzyme: || 1uL || (add last; it's the most expensive ingredient) | + | |Each enzyme: || align="left" | 1uL || (add last; it's the most expensive ingredient) |
|- | |- | ||
| - | |Sterile water: || ~25uL || (so that total volume is 50uL) | + | |Sterile water: || align="left" | ~25uL || (so that total volume is 50uL) |
|} | |} | ||
Revision as of 17:54, 22 July 2010
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Digestion Recipe
| DNA: | 12uL | |
| 10x BSA: | 5uL | (if needed, check NEB website) |
| 10x NEBuffer 2: | 5uL | (works with E, X, S, P) |
| Each enzyme: | 1uL | (add last; it's the most expensive ingredient) |
| Sterile water: | ~25uL | (so that total volume is 50uL) |
Ligation Recipe
Electroporation Transformation
Cells: E. coli DH10B
Protocol:
- Get DNA (~10uL?)
- 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 Eppendorf tube
- Incubate cells at 37oC for 30 to 60 minutes. Warm up agar plates in anticipation.
- Centrifube 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.
