blunt end ligation and peg 8000

Hiranya Roychowdhury hroychow at NMSU.EDU
Wed Jul 30 16:29:09 EST 1997


At 11:24 AM 7/30/97 +0200, leclair wrote:
>Hi, all,
>
>I am trying to perform a quite tricky blunt end adapter ligation to
>double stranded cDNA molecules. Unfortunately the ligation efficiency
>following the protocol is not good enough. The amount of the leftover
>sample does not allow me to increase the concentration of the cDNA but I
>could increase the amount of adaptors. 
>When I looked up blunt ended ligation in several molecular cloning books
>I found that the addition of PEG 8000 is said to improve ligation
>efficiency. Alas, all three books gave different concentrations for the
>PEG 8000 ranging from 5% to 15%. They also gave different factors of
>improvement ranging from 3 fold to 1000 fold.
>
>Does anybody have experience with the addition of PEG 8000 to blunt end
>ligation reactions or could anybody point out other ways of improving
>it?
>
>Thanks a lot					Frank
>
>

I routinely do blunt-end ligation with the so-called "Focus" buffer in 10 uL
volumes: 50mM Tris.Cl, pH 7.6, 10 mM MgCl2, 1mM ATP, 1mM DTT, 5% PEG 8000. A
5x stock may be stored in aliquotes at -20 or -70 C for years. This buffer
works fine with linkers/adaptors. The ligation, in my hands, are usually
over by 1 to 2 hrs at room temp. The original Focus study did comparisons
with different PEG conc. and found 5% to be the optimal at 22-24 C for 5hrs.
The PEG conc. in the transformation should not exceed 0.5%. Normally, I
transform 50 to 100 uL cells with 2 to 3uL of the ligation mix.

Hope this helps.




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