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