Team:Stanford/Protocols

Digestion Recipe

Ligation Recipe

Electroporation Transformation

Cells: E. coli DH10B

Protocol:

1. Get DNA (~10uL?)
2. Transfer DNA to electrocompetent cells (keep cells in ice bucket)
3. Resuspend cells and DNA
4. Transfer cells and DNA to cuvette (don't leave air bubbles, and hold cuvette at the top to keep the bottom cool)
5. Zap the cells (2000V, 25uF, 200ohms)
6. Add 1000uL SOC to cuvette, resuspend the cells, and transfer to Eppendorf tube
7. Incubate cells at 37oC for 30 to 60 minutes. Warm up agar plates in anticipation.
8. Centrifube the cells, remove 800uL of supernatant, resuspend cells in remaining 200uL.
9. Plate the cells, incubate at 37oC for 12-16 hours.

Heat-Shock Transformation

Cells: E. coli DH5alpha

Protocol:

1. Take out competent E.coli cells from –80 C freezer.
2. Turn on water bath to 42 C.
3. 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.
4. Keep tubes on ice.
5. Add 50 ng or 2.5 ul of circular DNA into E.coli cells. Incubate on ice for 30 min to thaw competent cells.
6. Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
7. Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
8. Add 1 ml of SOC. Incubate tubes for 2 hours at 37 C.
9. Spread about 100 ul of the resulting culture on LB plates with the appropriate antibiotic. Grow overnight.
10. Pick the colonies about 12-16 hours later.