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Sailesh Surapureddi Sailesh.Surapureddi at mcb.liu.se
Wed Aug 7 09:54:59 EST 1996



Hi  Everybody

        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
btw.

So pls feel free to shoot if you have any more Qs.

Good luck.

Sailesh.

Dr.Sailesh Surapureddi
Plan 12; Inst. for Cellbiologi
Halsouniversitetet
S 581 85, Linkoping
Sweden.

Tel: +46-13-223917 (W) +46-13-138839 (R)
Fax: +46-13-224149
E.mail: Saisumcb at hulio.liu.se
        Saisu at mcb.liu.se





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