Bio-Rad Ligation and Transformation Module - Manual (Page 47)

9. Resuspend the bacterial pellet in 300 µl of ice-cold transformation
buffer by gently pipetting up and down in the solution above the pellet
with a 1,000 µl pipet, and gradually wear away the pellet from the bottom
of the tube. Make sure that the bacteria are fully resuspended, with no
clumps. Avoid removing the cells from the ice bucket for more than a
few seconds.
10. Incubate the resuspended bacteria on ice for 5 min.
11. Centrifuge the bacteria in a microcentrifuge for 1 min. Note: Ensure
that the bacteria are on ice immediately prior to and immediately
following centrifugation. If the centrifuge is not close to the laboratory
bench, take the entire ice bucket to the microcentrifuge so that the
bacteria are only out of the ice bucket for 1 min. Use a refrigerated
microcentrifuge, if available.
12. Remove the supernatant from the pellet using a 1,000 µl pipet or
vacuum source.
13. Resuspend the bacterial pellet in 120 µl of ice-cold transformation
buffer by gently pipetting up and down with a 200 µl pipet. Be sure that
bacteria are fully resuspended with no clumps. Avoid removing the
cells from the ice bucket for more than a few seconds.
14. Incubate the resuspended bacteria on ice for 5 min.
The cells are now competent for transformation.
Experimental Procedure for Transformation
15. Label one microcentrifuge tube with your initials, insert name, and
"TF" (referred to below as the "gene TF" tube).
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