BclI and BamHI ligation
Dennis J. Templeton
djt2 at po.CWRU.Edu
Tue Sep 29 10:18:35 EST 1992
Previously, on this channel...
>> Call me paranoid...but EVERYTIME I alkaline phosphatased a
>> vector, then gene-cleaned it, the ligations never worked. NOTHING!
>> If I purify the fragment by gene-clean, then phosphatased it and
>> precipitated it by traditional means, the ligations always work!
>> I don't lnow why, but it appears that phosphatased, then gene-
>> cleaned vector doesn't work in my ligations. Therefore I never
>> prepare my vector that way anymore.
>> Anne Howe
We have had good luck usin cip and home-made gene clean this way:
Do your digest, 37C, 60 minutes or so using 1 ug vector
Add 1ul cip (Boerhinger 1u/ul) for 5' ends or 5-10 u for blunts
37C for 15-20 minutes right in the RE buffer
Run the cut vector out on a gel, usually on the same gel as the insert prep
Cut out the band and recover with NaI/glass
ligate about 25-50 ng vector with a 10x excess of insert
This has two advantages:
1) if your digest is incomplete, you will both know it and remove
the uncut vector
2) the gel + glass powder elution is good at killing the cip
And it's easy!
p.s. to Anne at Glaxo... I'll trade tips for stock anyday.
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