Team:Calgary/Notebook/ProtocolManual/DNA
From 2014.igem.org
DNA Protocols
Preparing Chemically Competent E. coli Cells
- Inoculate 5-10mL LB with Top10 E. coli culture at 37oC shaking over night
- Subculture 1mL of bacteria into 50mL LB at 37oC shaking until OD600 is 0.4-0.6 (~2.5 hr)
- Centrifuge the subculture at max for 20 min at 4oC
- Resuspend pellet in 12.5mL cold CaCl2 (50mM, 15% glycerol)
- Add 10μL of ddH2O to appropriate well [will turn orange]
- Incubate at RT for 10 min
- Use 1μL of DNA to transform cells Store plates in -20oC
- Thaw 100μL of competent cells on ice right before use (do not thaw completely)
- Add DNA (max 20μL) to cells, flick to mix, and ice for 30 min
- Heat shock 2 min at 42°C
- Ice 5 min
- Add 250μL LB medium to each tube
- Incubate 30-60 min at 37°C shaking (Kan is always 1h+)
- Spin down (14,000 rpm for 2 min) and remove all supernatant except ~100μL
- Resuspend, spread 50μL on antibiotic plate Grow overnight in incubator at 37°C
Rehydrating Registry DNA
Bacterial Transformation