problem cloning a PCR amp. fragment

Jill Kellogg jkellogg at
Tue Jun 18 11:13:28 EST 1996

On 6/16/96 Anwar Khan wrote:
>I am trying to clone a 200bp PCR amplified fragment containing
>EcoRI and BamHI sites in the primers (vector is pGEX).  I get positive
>clones but none of them have inserts.  I have tried up to    1:10 ratio of
>vector to insert.  Any ideas what is wrong?  

It could be that EcoRI and BamHI are not efficiently cutting the sites in the
PCR fragment thus reducing the number of available fragments for ligating.  If
you have access to a TA cloning vector it would be worth trying.  You could
always then cut it out of the TA cloning vector with EcoRI and BamHI if you need
to move it somewhere else.

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