PCR cloning

Richard Daines rdaines at ctol.net
Mon Jul 21 17:01:34 EST 1997


On Mon, 21 Jul 1997 12:47:53 -0600, dlwheele at blue.weeg.uiowa.edu
wrote:

>Hello.  I am trying to clone a 6kb PCR fragment into a 5 KB
>plasmid and have had no success.    I have cloned restriction
>sites (Kpn I and Xho I) into my PCR product and verified them
>by
>sequencing.  I have put four extra bases (GCGC) on the end of
>the Kpn I site and six extra bases (GCGCGT) at the end of the
>Xho I
>site. 
>	
>Thanks in advance,
>
>
>Deric Wheeler
>
>dlwheele at blue.weeg.uiowa.edu
>
>
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While I don't have the NEB catalog in front of me, I might venture a
guess that one or more of your restriction site(s) on the PCR product
are not cutting due to being too few  nucleotides from the end.  You
might want to try using a TA vector (provided you PCR product has the
correct one base overhangs).  I have had very good results with the
kit from Invitrogen.  One a plasmid is recovered, try those cuts to
recover the fragment.

Richard J. Daines
Mystic, CT

rdaines at ctol.net
http://www.ctol.net/~rdaines/
NAR# 70562 SR



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