Bio-Rad Ligation and Transformation Module - Manual (Page 33)

7. Centrifuge at top speed for one
minute and immediately put tube
on ice.
8. Use a 1000 µl pipet or a vacuum
source to remove culture
supernatant avoiding the pellet.
Keep the tube on ice.
9. Resuspend the bacterial pellet in
300 µl of ice cold transformation
buffer by very gently pipetting up
and down in the solution above
the pellet – do not touch the pellet.
10. Incubate the resuspended bacteria
on ice for 5 min.
11. Centrifuge the bacteria at top
speed for 1 min. Ensure the
bacteria are on ice immediately
prior to and immediately following
centrifugation.
12. Using a 1000 µl pipet or a vacuum
source, remove the supernatant
avoiding the bacterial pellet.
13. Very gently resuspend the
bacterial pellet in 120 µl of ice
cold transformation buffer. Keep
tube on ice.
14. Incubate resuspended bacteria
on ice for 5 min. The cells are
now competent for transformation.
29
Ice
TF buffer
TF buffer
Ice
Ice