TITLE: CK111 comp cells electroporation
AUTHORS: Elizabeth Fozo

[STEPS]
?. [How to generate competent CK111/pCF10-101 cells for transformation]
In 10 mL BHI with 100 µg/mL erythromycin, start an overnight culture of EC1000 containing your plasmid of interest.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
In 10 mL BHI with 1 mg/mL spectinomycin, start an overnight culture of CK111/pCF10-101.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Next morning, dilute the overnight culture of CK111 10-fold after determining the amount of culture that will be required (25 mL for 2 transformations).
Rachel suggests that the pCF10-101 plasmid is stable; adding spectinomycin is unnecessary.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Grow cells at 37°C until OD600 is between 0.5 and 1.0. Suggest 0.75. This takes ~2 hours.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Isolate plasmid of interest from the overnight culture of EC1000 containing your plasmid of interest.
Rachel suggests that the plasmid isolation does not need to be done the same day; can be done at an earlier time.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Once CK111 has reached the proper OD600, aliquot your cultures into 25 mL conicals which have been chilled on ice.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Chill cells on ice for 15-20 minutes.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Pellet cells at 3500 RPM at 4°C for 10 minutes.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Pour off supernatant and wash with cold water. Spin again.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Resuspend each pellet in 500µL lysozyme solution containing 30µg/mL lysozyme. Add lysozyme to the solution fresh just before use. Leave cells in the same conical.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Incubate cells for 20 min at 37°C.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Flood cells with cold water. Wash two times.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Resuspend pellet in 800µL of electroporation solution, and transfer to a chilled Eppendorf tube. Spin cells at 13K RPM for 3 minutes at 4°C.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Resuspend the pellet in 85µL electroporation solution.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Transfer 50µL of these competent cells to a new Eppendorf tube.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
To your competent cells, add 50 ng, 100 ng, or 150 ng DNA (this is variable; attempt different concentrations).
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Add cells to the prechilled electrocurvette and use the EC2 settings on the electroporator.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
After electroporation, rescue cells in 500µL plain BHI for 2 hours using a snap cap tube. Incubate statically or shaking at 100 RPM at 37°C.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Transfer 100-125µL cells to a pre-warmed BHI plate containing 10µg/mL ERM and 250µg/mL X-gal.
Rachel suggests plate 100µLof cells on 5 plates; do NOT spin cells down to concentrate. It is not many plates and eliminates the difficulty of resuspending the cells (it is very difficult after lysozyme treatment and electroporation).
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Incubate plates for 24-48 hours and look for blue colonies.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Restreak blue colonies on the same selective media. Freeze good clones.
?. [How to generate competent CK111/pCF10-101 cells for transformation]
Optional: isolate plasmid from CK111 and do a PCR to ensure the correct plasmid is in the cells.