checking ligations by PCR, help.
un691cs at genius.embnet.dkfz-heidelberg.de
Mon Nov 25 11:10:41 EST 1996
In article <56sumt$oqu at fu-berlin.de>, hoege at chemie.fu-berlin.de says...
>I want to test ligations by PCR, since I used the shotgun approach to
>clone a 1.3 kb fragment, that is in pUC18, into the binary vector
>bin19. I linearised bin19, filled in the cohesive ends and made a
>blunt end ligation with the filled in fragment of pUC18. When I
>transform these, I cannot select for the insert...so I first want to
>check the ligation by PCR with a Bin 19 and a insert primer.
>Has anyone experience with PCR of ligations?
yeah. it doesn't work.
(mostly because the vector specific primer will anneal to
empty or religated vectors, and you get an enormous background
of low-weight 'Ampli-Schmutz')
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