HELP! ligation

Chaitanya Athale chaitanya.athale at ibc.unibe.ch
Fri Sep 25 05:26:37 EST 1998


dear Users,
thanx for all the ideas. 
the details of my reacn were,
- 5-6 bps beyond the restr site towards the ends of the fragment
-PCR fragment size separated and purified
-plasmid restr enz cut, purified, and dephosphorrylated
-ligation at 16 deg C, o/n
-transformation with clean dna 6x1^5 effic (per ug)
-ligation product seen on gel, but no transformants in the plates!
- a paper in Anal. biochem recommends heat ncativation of ligase (70deg C,
15 min) to increase efficiency 260-fold!! Will be doing this!

any more ideas or opinions on this?

Sincerely,
Chaitanya
 
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Chaitanya A. Athale,
Dept. of Chemistry and Biochemistry,
Universität Bern,
Freiestrasse 3,
CH-3012 Berne,
Switzerland.

Tel.:  ++41/ (0) 31/ 631 4337
Fax: ++41/ (0)31/ 631 4887
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