Problems with TA cloning

[Haley Lindsey]

I'm trying to clone a piece of PCR amplified DNA into a vector using an
Invitrogen TOPO TA cloning kit that a rotating grad left behind. I planning
to use phusion for my actual reaction so I tried amplifying the kit control
PCR reaction with phusion, using Qiagen PCR purification afterwards and then
incubating the purified product (30uL) with 3uL 10x buffer, 1uL of Taq and
1uL of 10mM dNTPs for 20 minutes at 72C to add A overhangs. I also amplified
the control DNA fragment with Taq as a check. When I run these reactions out
on a gel I am seeing clear bands at the right place.

I then cloned the DNA into the provided vector according to the kit
instructions and did a transformation, plating on Kan (the control fragment
is a Kan resistance cassette). I got nada for both the Taq and the Phusion
with Taq extension reactions. I don't think my competent cells are the
problem as my positive control reaction (PBR322 on tet plates) yielded
colonies. I think the cloning step is probably the issue. Any ideas on what
I'm doing wrong?

-Haley, University of Washington

[Emerson A. Castilho Martins]

I think that the correct control for transformation is some circular vector
that offers kan resistance, because if you have plates with wrong [kan], pbr322
will not show you.

On Quarta-feira 09 Dezembro 2009 17:19:49 Haley Lindsey wrote:
> with Taq extension reactions. I don't think my competent cells are the
> problem as my positive control reaction (PBR322 on tet plates) yielded
> colonies. I think the cloning step is probably the issue. Any ideas on what
> I'm doing wrong?
>
> -Haley, University of Washington

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--
*********************************
Emerson A. Castilho Martins
Doutorando em Fisiologia Geral
Depto. de Fisiologia Geral
Instituto de Bioci�ncias
Universidade de S�o Paulo
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