Time: 2.25 hr
- Competent cells
- Antibiotic LB plates
- Deactivate the ligase by heating at 65 degrees for 20 minutes.
- Chill ligated DNA on ice
- Thaw competent cells ((found in eppendorfs in box on second-shelf down of -80ºC freezer) on ice for ~5 min.
- Add 3 μL ligation mixture to cells in bottom of tube
- Incubate on ice for 30 min
- Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).
- Incubate on ice for 2 min
- Add 500 μL warm SOC media
- Shake in 37ºC room for 1 hr
- Spread 200 μL on LB/antibiotic plates using rotator and glass spreading wand.
- Incubate plates in 37ºC room overnight.
1To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)