pGEM-T easy vector gives extra band
SURESH KUMAR K.G.
skg at biochem.iisc.ernet.in
Wed Mar 7 13:04:34 EST 2001
Prepping yourself means, you transform strains like JM109/XL1-Blue
with your vector (pGEMT), pick single colonies, do mini/midi/maxi preps,
You will have more than enough vector for a life time...
Hope it is clear..
On 7 Mar 2001, R. Jayakumar wrote:
> 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
> ----- 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?
> > Susanne
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