From Powers Lab UC Davis
1. Remove competent cells from -80°C
freezer (already aliquoted into 50 µl volumes in 1.5 ml microfuge tubes).
Thaw on wet ice.
2. Gently mix cells by stirring with the end of a yellow pipette tip.
3. Add 1-3 µl of DNA directly to the tube containing the competent cells
moving the pipette tip through the cells while dispensing. Gently tap tube to
mix.
4. Incubate cells on ice for 30 minutes.
5. Heat shock cells for 20-30 seconds at 37°C.
6. Place on ice 2 minutes.
7. Add 950 µl of room temperature LB or SOC medium.
8. Shake at 225 rpm for 1 hour at 37°C.
9. Spin down cells at maximum speed (14,000 rpm in microcentrifuge) for 30 seconds.
10. Remove supernatant (the media).
11. Add 100 µl LB. Resuspend very gently.
12. Plate 95 and 5 µl onto LB + appropriate antibiotic. Add 100 µl
medium to the 5 µl aliquot to allow spreading on the plate.
13. Add 10-12 glass beads to the plate.
14. Roll the beads over the agar until the cells are fairly evenly distributed.
15. Incubate overnight at 37°C.