Emergent-----Cloning problem

Wolfgang Schechinger hubahopp at gmx.de
Fri Nov 22 05:48:52 EST 2002


Daer HaoJiang,

you need to give us some details about the design of your construct and the
other ingredients involved in your experiment.

Are all these plasmids/inserts you get in the minipreps similar identical
in size? Can you recover your insert with restriction enzymes, i.e. are you
loosing some sequence from the vector or the insert? Can you get any
evidence from sequencing?
The only way to solve your problem might be to stepwise dissect your
cloning / ligation system and use lots of appropriate controls including
control inserts and control vectors.

It's almost impossible to give you any solution now since every step
performed could bare the reason for not working (recombination effects,
contamination, logical error in experiment design, bad or wrong labelled
enzymes, bad Fung Shui for position of reaction vessels including undesired
effects of the moon phase (after some years in this business, one becomes a
little sarcastic. Sometimes) One simply cannot find any reason for
experiments to fail and if you do it again, (or again and again, RE-search
is named what we are doing (hehe, hi Susanne :). Preferably you have
finished all your reagents and need to use all completely new vials and
then suddenly everything is fine, of course without any obvious reason...

You also should check this NGs archive at http://www.bio.net or or
http://groups.google.com, in the past months we had several discussions on
similar problems. 

Good luck!

Wo

At 12:05 PM 2002/11/21 -0800, Haojiang wrote:
>Hi, Dear all.
>
>I am making a construct for mice knockout. After the ligation of the
>insert and the vector and transfect into stable II cell, I got plenty
>of colonies. But when I screen the colonies, most of them is smaller
>in size than the vector! I had tried this for quite a few times, but I
>can not get it. Anyone please give me some hints?!!!!
>
>Thanks!
>
>

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