Sailesh.Surapureddi at mcb.liu.se
Wed Aug 7 09:54:59 EST 1996
Here is the ice cold transformation protocol.. I am posting this
for the benifit all transformers.
1. Pre warm the plates @37 (very imp) for 30min or 60min which ever is
convient to you.
2. Chill the tranformation tubes (falcon 2059 or clean eppies, the later
takes more time to chill)
3. Thaw your competent cell on ice waterbath.
4. Open them on ice only and aliquot to the pre chilled tubes (if they are
from Life tech, I usually take 25ul per tube).
5. Add the DNA, (if its ligated DNA, make sure to clean it with ethanol
pption and compeltely dry it).
6. I routinely take 2-4uL of DNA (or ligated mix i.e 1/4th of the 20ul
ligation mix) for 25 -50uL of competent cells.
7. Add slowly with gentle swirl, with the pipette tip.
8. Allow it to stay on ice for 3-5min.
9. Take the entire amount and spread on the prewarmed LB +antibiotic
plates (no SOC etc etc).
10. Incubate for 10-12hrs @37C.
Low! you have your tranformations with the higest effiencies you have seen
so far. If you are using some DNA, make sure its clean. Ofcourse! for me,
it worked with even the yeast shuttle vectors. They were crude preparations
So pls feel free to shoot if you have any more Qs.
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