T4-pol PstI/Exo EcoRI/fill in

Ricky Boernke ricky_boernke at gmx.net
Tue Oct 30 14:43:44 EST 2001


Tom Knight <tk at suspiria.ai.mit.edu> wrote in message news:<vuy1yjlnpsq.fsf at suspiria.ai.mit.edu>...
> Have you considered making a pair of primers containing the exact
> restriction site you'd like to see, and doing PCR around the vector,
> followed by a cut and religation?  I've found that to be a more
> reliable approach, in general.
> 
> Ricky Boernke <ricky_boernke at gmx.net> writes:
> > I would like to remove all restriction sites but the HindIII site from
> > the pUC18 polylinker region.

Hi Tom,

the T4 approach didn't work I came up with only 4 colonies which turned out
to be something but pUC. So I will try to take the PCR approach. Couldn't I
also use 5' phosphorylated oligos, DpnI digest the parental DNA and just
ligate the PCR product and transform into E. coli?

Ricky




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