pGEM-T easy vector gives extra band
jakku at mrna.tn.nic.in
Wed Mar 7 10:55:51 EST 2001
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
thank you for your helpful suggestion
----- 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
> > to buy a new stock.
> uhm, how about prepping some yourself?
More information about the Methods