No ligation of PCR product in pGemT-Easy

Jayakumar, R R.Jayakumar at RoswellPark.org
Wed Jul 30 08:58:38 EST 2003


That is interesting.  I never considered phosphorylating PCR products =
when using pGEMT vectors.  The cloning was too good anyway.. so why =
bother.   Anyway, thank you for that useful information.   My apologies =
for my comments earlier.
Jayakumar



-----Original Message-----
From:	"Dr. Hiranya S. Roychowdhury" [mailto:hroychow at nmsu.edu]
Sent:	Tue 7/29/2003 5:54 PM
To:	methods at hgmp.mrc.ac.uk
Cc:=09
Subject:	RE: No ligation of PCR product in pGemT-Easy


Interesting!  I really do know what "TA-cloning" means; but the presence =
of=20
T-overhang and having to phosphorylate the amplified product are not =
related so=20
far as the "clonability" issue goes. The idea of having a T-overhang has =
little=20
to do with phosphorylation state and its bearing on ligation reaction. =
But you=20
are right in pointing out that a certain percentage of self-ligation of =
the=20
vector (both intra- and intermolecular) may pose a problem in some cases =
(if=20
the vectror itself is phosphrylated).  I suppose my personal observation =
of=20
phosphorylated products fairing better may have to do with the fact that =
the=20
two phosphorylated ends ligate far more easily, thereby limiting =
self-ligated=20
products.

BTW, I am not sure that the vectors come phophorylated (as suggested by=20
Duncan).

Hiranya

Quoting "Jayakumar, R" <R.Jayakumar at RoswellPark.org>:

> Shouldn't need to.. that is the whole idea of  T-overhang cloning.   =
Only the
> PCR products have the A-overhang which sits into the pGEM-T cloning =
site.=20
> Sometimes, I have seen some amount of self-ligation may happen within =
the
> vector.. which might have been the case with joe.=20
> =20
> jayakumar
>=20
>=20
> -----Original Message-----
> From:	"Dr. Hiranya S. Roychowdhury" [mailto:hroychow at nmsu.edu]
> Sent:	Mon 7/28/2003 7:02 PM
> To:	methods at hgmp.mrc.ac.uk
> Cc:=09
> Subject:	Re: No ligation of PCR product in pGemT-Easy
>=20
> There is one basic thing you may have overlooked!  Did you =
phophorylate the
>=20
> primers (prior to PCR) or the amplified products before ligation?
>=20
>=20
> Quoting Joe <appelgjj at sci.uovs.ac.za>:
>=20
> > Hi,=20
> >=20
> > I've obtained several clones by means of differential display, where
> > after I've reamplified the clones, with Taq SuperPak =
(Sigma-Aldrich),=20
> > cleaned the PCR product by means of GFX PCR DNA purification kit
> > (Amersham Biosciences) and ligated the purified clones into =
pGemT-Easy
> > (Promega) and transformed into JM109 cells.  The transformed cells
> > were grown on LB + 50mg/ml Ampicillin.  I've got cells that grew,
> > extracted plasmid again with a miniprep, and digested with EcoR1, =
but
> > got no insertions back.  I then plated the transformed cells on
> > Xgal/IPTG plates and everyone was blue - meaning no recombination
> > occurred.  Can anybody please help?
> >=20
> > Thank you in advance
> >=20
> > Joe Appelgryn
> > M.Sc student
> > Dept. of Plant Sciences
> > University of the Free State
> > RSA
> >=20
>=20
>=20
> --=20
> Hiranya S. Roychowdhury, Ph.D.
> Coll. Asst. Professor,
> Molecular Biology,
> Dept. of Chemistry & Biochemistry
> Rm# 336, Chemistry Bldg.; MSC 3MLS
> New Mexico State University
> Las Cruces, NM 88003
> ---
>=20
>=20
>=20
>=20


--=20
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---



---



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