1. Chill sterile microfuge tubes or 17 x 100 mm polypropylene culture tubes (use the latter when doing this after ligation)
2. Thaw competent cells by placing them on ice for 5 minutes, and treat them gently as they are fragile.
3. Keep on ice and don't let them sit for long. Gently transfer thawed cells to chilled tubes.
4. You can use 50 - 100 μL of cells. Add DNA to cells and gently flick the mixture.
5. Incubate on ice for 10 minutes.
6. Heat shock cells for exactly 45 seconds in water that is exactly 42° C. Immediately, go to step 7 below. Have the ice next to the water bath to minimize the delay between steps 6 and 7.
7. Place on ice for 2 minutes.
8. Add 900 μL of LB no amp (or other antibiotic). Let cells recover for 1 hour in 37° C incubator, no shaking. After this, the cells are no longer fragile.
9. Spin cells, decant medium, add 100 μL of LB plus amp (or other antibiotic). Resuspend pellet and pipet 90 μL and/or 10 μL of cells onto LB amp plates (or other antibiotic).
10. Place plates in 37° C incubator overnight (10 - 16 hours, max.). Pick colony that is not near other colony and watch out for satellite colonies.
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