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
>> Glaxo
>> 

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!

trust me.

p.s. to Anne at Glaxo... I'll trade tips for stock anyday.

dennis



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