pGEM-T easy vector gives extra band

R. Jayakumar jakku at mrna.tn.nic.in
Wed Mar 7 10:55:51 EST 2001


DEar Susanne
      Can you please explaing what you meant by the answer.  it will be
helpfull.  actually, we got pgemt from promega.  I know that it is possible
to generate T tailed vectors.  I also have some papers from NAR.  But have
you done this in your lab.  if so can you send me the standardised protocol
for that.  I will be very grateful for your lab.
    Anyway, the insert was flanked by the two sites HindIII/PstI inserted on
either sides through the primers which was synthesised with these sites at
the 5' end, for a specific purpose.  The insert does get released as
expected when idigest the chimeric plasmid with these enzymes, but for the
extra band.
   thank you for your helpful suggestion
sincerely
jakku

----- Original Message -----
From: "Susanne Rohrer" <srohrer.nospam at immv.unizh.ch>
To: <methods at hgmp.mrc.ac.uk>
Sent: Wednesday, March 07, 2001 1:34 PM
Subject: Re: pGEM-T easy vector gives extra band


>
>
> "R. Jayakumar" wrote:
>
> > hi..
> >    I have these PCR fragment of 200bp in a pGEMT easy vector in JM109
> > strain.  After digesting it with HindIII/pstI , I get an additional 1 kb
> > fragment other than my linearised vector and the 200bp insert.
>
> Doesn't that appear to be the terminally supercoiled plasmids that are not
> digestible? There have been several discussions about that on this NG.
>
> >  I have not tried fresh ligation with fresh pGEMT again, just
> > because, we are running very much low on pGEMT and do not have enough
money
> > to buy a new stock.
>
> uhm, how about prepping some yourself?
>
> Susanne
>
>
>


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