checking ligations by PCR, help.

slavemaster z 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...
>
>Hello there!
>
>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.

z



(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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