cloning problems

Dr A.H.M. Van Vliet AVV2 at le.ac.uk
Fri Dec 13 14:41:31 EST 1996


thees at mzdmza.zdv.uni-mainz.de wrote:
>Hi,
>this is Rudi from Mainz/Germany. I have a problem with cloning a cDNA
>clone into a pGST Vektor. Because of different pGST- and "cDNA clone
>Vektor"- polylinker it is only possible to ligate the clone with EcoRI; a
>site directed ligation is not possible. My problem is now that all of my
>inserts are in the false direction. So is there anyone with the same
>ligation problem or does anybody knows a solution to this problem? 
>
>email  thees at mzdmza.zdv.uni-mainz.de

Hi Rudi,

it can be a problem if your protein is toxic to the E. coli when cloned
in the right orientation in pGST. I don't know this pGST-vector, but if
it uses the lac/tac promoter like lots of other expression systems, then
there is always "leaking" of transcription, thus giving low expression of
fusion protein, which can be lethal to your E. coli's.

For you cloning woes, why not amplify your insert with specific primers which
have other (different) restriction sites added to them at the 5'-end, plus a 3
to 4 basepair clamp. Then you could do a directional cloning. Probably cheaper
to order to primers then retrying the cloning...

hope this helps,

Arnoud




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