Transforming chemically competent cells (Inoue) protocol
- TB buffer cells
- SOC stored at room temperature
- plate made with appropriate antibiotic
- Sterile 1.5-ml microcentrifuge tubes
- Measure out 25 - 200 µl of TB buffer cells into sterile 1.5-ml microcentrifuge tube (1).
Allow TB buffer cells to thaw on ice.
Do not use glass tubes which adsorb DNA.
- Measure out DNA into sterile 1.5-ml microcentrifuge tube (1).
Mix solution by pipetting up and down several times.
Keep volume of DNA less than 5% of the cell volume.
- Incubate on ice for 30 mins.
Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
- Incubate at 42°C for 30 secs.
- Incubate on ice for 2 mins.
- Add 4 volumes SOC.
- Incubate at 37°C for 1 hr with shaking at 200 rpm.
Note: Can also save some time here by reducing incubation to ~45 min.
Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics.
- Plate out 100 - 300 µl of DNA onto plate made with appropriate antibiotic.
- Incubate at 37°C for 12 hrs(overnight).
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 32 mins