pGEM-T easy vector gives extra band

R. Jayakumar jakku at mrna.tn.nic.in
Thu Mar 8 02:40:25 EST 2001


DEar Suresh
  that is not what she meant.  Moreover it not possible to amplify pGEMT in
the manner you say.   Since they are T-tailed vectors, first of all, it is
not possible to ligate a T-tailed vector until you have a PCR insert (which
is naturally flanked by A overhangs) in MCS region. Even if you manage to
blunt end ligate the pGEMT vector after shaving off the T-tail, you have no
means of regenerating the T-tail except by experimentally T-tailing it with
DNA polymerase and an excess of TTP, which I can very well do in some other
vector too.    it is this protocol which I wanted.
    Anyway, thanks a lot for your response
sincerely
jayakumar

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R. JAYAKUMAR
CSIR- Senior Research Fellow
Dept. of Molecular Microbiology,
School of Biotechnology,
Madurai Kamaraj University,
Madurai - 625 021.
India
email: jakku at linuxfan.com
tel: +91-452-858471-374
efax: (603)-688-4665
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"Life is a comedy for those who think and a tragedy for those who
feel" --Emerson
----- Original Message -----
From: "SURESH KUMAR K.G." <skg at biochem.iisc.ernet.in>
To: "R. Jayakumar" <jakku at mrna.tn.nic.in>
Cc: <methods at hgmp.mrc.ac.uk>
Sent: Wednesday, March 07, 2001 11:36 PM
Subject: Re: pGEM-T easy vector gives extra band


>
>
> Hi,
>
> 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..
>
> Suresh
>
>
> 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
> > 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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