DNA Protocols

Preparing Chemically Competent E. coli Cells

1. Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night
2. Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD₆₀₀ is 0.4-0.6 (~2.5 hr)
3. Centrifuge the subculture at max for 20 min at 4oC
4. Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)

Rehydrating Registry DNA

1. Add 10μL of ddH2O to appropriate well [will turn orange]
2. Incubate at RT for 10 min
3. Use 1μL of DNA to transform cells
Store plates in -20oC

Bacterial Transformation

1. Thaw 100μL of competent cells on ice right before use (do not thaw completely)
2. Add DNA (max 20μL) to cells, flick to mix, and ice for 30 min
3. Heat shock 2 min at 42°C
4. Ice 5 min
5. Add 250μL LB medium to each tube
6. Incubate 30-60 min at 37°C shaking (Kan is always 1h+)
7. Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL
8. Resuspend, spread 50μL on antibiotic plate
Grow overnight in incubator at 37°C