HELP: Cloning PCR products

Joe Makkerh WCI jm144 at mole.bio.cam.ac.uk
Tue Apr 25 11:33:16 EST 1995


sl6dp at cc.usu.edu writes:

>I am trying to clone some PCR products resulting from an anchored-PCR
>(oligo dG tailing of 3' ends and use of an oligo dC primer with restriction
>sites). The product consists of some bands and quite a lot of smear 
>between them, resulting from the unespecific atachment of the 3'-end primer.
>After size-selection of a gel, purification and restriction digestion of the 
>products, I ligate them to the vector, also cut with the same 2 enzymes 
>(directional, sticky-ends cloning). The product seems to be cutting (after
>running a gel it seems a little bit lower), the vector is also cutting fine,
>but it doesn't seem to be ligating. Do you think I should try using 
>adaptors or linkers. Do you have any experience with this kind of problem?
>Any help will be very welcome!!!!!!!!!


I have recently started to use the TA cloning kit from Invitrogen. It seems
to work very well, but requires an extra day over direct cloning. I'll post more
detials if you're not familiar with the system, but look in any invitrogen
manual if you've got one handy. Recently, the way they generate the TA vector
has been made accessible to everyone, but not by Invitrogen themselves.

Joe



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