RbCa competent cell preparation

Materials required

10X RbCa TXN salt

chilled autoclaved MQ

sterile SS34 oakridge tubes/50ml falcons

sterile 30% glycerol

Method

· use 1ml o/n grown culture as primary inoculum in 25ml of 2X LB/ rich media

· grow at 200rpm, 37 degree Celsius till OD reaches 0.4-0.6

· transfer the culture in 50ml falcons and stand the culture in ice for 10min. Simultaneously pre-cool the centrifuge to 4 degree. (use SS34 tubes preferably)

· harvest the cells at 4000rpm, 10min, 4 degree.

· decant the supernatant, wash and resuspend the pellet in 15ml 1XRbCa.

· stand it in ice for 15 min.

· spin the cells at 4000rpm, 4 degree, 10mins.

· decant the supernatant and resuspend the cells in 2ml of 1xRbCa.

· leave it at 4 degree for 24 hrs to increase the efficiency.

Storage

· prefer using fresh.

· to store at -80 add final 15% glycerol, snap chill with liquid nitrogen and store

· avoid using beyond 2 months.