Cloning problems-Help!!

Jarmo Schrader Jarmo.Schrader.removethis at genfys.slu.se
Mon Nov 29 03:38:11 EST 1999


Alexander,

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.

Jarmo


In article <3841655C.A7E7D861 at nefo.med.uni-muenchen.de>, zimprich
<zimprich at nefo.med.uni-muenchen.de> wrote:

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> 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
> coloumns.
>

-- 
Jarmo Schrader     Umea, Sweden

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