Jarmo.Schrader.removethis at genfys.slu.se
Mon Nov 29 03:38:11 EST 1999
could it be that your insert codes for a protein that is lethal for E
coli? Do you use a vector with blue/white selection? If so, try plating on
plates without IPTG to prevent induction.
In article <3841655C.A7E7D861 at nefo.med.uni-muenchen.de>, zimprich
<zimprich at nefo.med.uni-muenchen.de> wrote:
> Content-Type: text/plain; charset=iso-8859-1
> Content-Transfer-Encoding: 8bit
> Dear Colleagues,
> In the last few months, i have a very big cloning problem,.
> I try to get a AOC1 (5´overhang CC!TGAGG), ECO R1 (also 5´overhang
> G!AATTC) fragment into a vector also cut with AOC1, ECO R1. It sounds
> quite easy, but it is a nightmare.
> I tried it over a dozen times, but i only get very few colonies after
> transformation, which were all wrong. I did all kinds of control
> experiments; so let us assume that Ligation, Transformation etc. do work
> in my hands.
> Is it possible the some restriction enzyme overhangs get lost, or need a
> special treatment for ligation?
> I purify my DNAs over agarose gels and clean them up using Quiagen
Jarmo Schrader Umea, Sweden
Before replying to my e-mail delete 'removethis.' from the address!
More information about the Methods