IGEM:PennState/2006/Transformation

Transformation

Time: 2.25 hr

Media/Reagents

• Competent cells
• SOC¹
• Antibiotic LB plates

Pre

• 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.

Transformation

1. Add 3 μL ligation mixture to cells in bottom of tube
2. Incubate on ice for 30 min
3. Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).
4. Incubate on ice for 2 min
5. Add 500 μL warm SOC media
6. Shake in 37ºC room for 1 hr
7. Spread 200 μL on LB/antibiotic plates using rotator and glass spreading wand.
8. 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)

Cloning

Main