- Grow E. coli strain ( JM101,NM522,DH5af', etc.)in 2X YT to A260 = 0.3; it is very important to use early log phase cultures for good transfection efficiencies. Different strains have different growth rates.
- Chill the culture in an ice bath before spinning out the cells (6K,7',4｡). pour off the sup. and wipe the bottle walls with a Kimwipeｪ and resuspend the cells in 2 ml of transformation buffer (TB) per 10 ml of original culture.
- Spin again 5K,7',4｡,in the HB4 rotor.
- Resuspend the pellet in 0.25 ml cold TB per 10 ml of original culture and hold on ice for 30'.
- Add 9 ul of DMSO (from a freshly opened bottle, it may also be frozen) for each 10 ml of original culture. Hold on ice for 10 min.
- Aliquots of 100 ul should be stored at -70｡ C for future transformations.*
- TRANSFECTION
- Add DNA to prepared cells ( no more than 1/10th volume) .
- Hold on ice for 40 min with occasional mixing.
- Heat shock the cells for 2 min. at 37｡C.
- Add 1 ml 2X YT broth and incubate at 37｡C for 40 min.
- Plate a small amount onto a drugged plate and grow overnight for the titer. Store the remainder at 4｡C.
- Cells will gradually become less competent as they sit frozen. They should be good for about 3-4 weeks.