Removing FRT-flanked Cassettes
- Grow cells in ~4 mL LB until cloudy (OD600=0.5)
- Put on ice
- Transfer 1mL into an eppendorf tube on ice, let cool
- Centrifuge full speed for 30 sec, toss out supernatant
- Resuspend in 90uL of TSS solution
- Add 10uL KCM
- Step 6.5: do the negative control before adding the DNA (plate 70uL of cells on amp. No cells should grow. This ensures that the cells aren't contaminated and that only the uptake of p21 will confer ampR)
- Add 1uL plasmid DNA
- Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute, rescue 1 hr, then incubate and/or plate
Always do negative controls on your competent cell prep!
Sector a region of your plate and spread about 5uL of untransformed competent cells to confirm that they are free of contamination.