Ligation problems

Nico Dantuma nico.dantuma at mtc.ki.se
Sun Aug 9 08:52:55 EST 1998


>Date: Sun, 09 Aug 1998 15:57:52 +0200
>To: tbu at gmx.net (Thomas Burmeister)
>From: Nico Dantuma <nico.dantuma at mtc.ki.se>
>Subject: Re: Ligation problems
>In-Reply-To: <9888213419.~INN-EQJa00189.bionet-news at dl.ac.uk>
>
>Hi Thomas,
>
>Are you sure it is the ligation step that is failing or is your
observation that you don't have colonies after the transformation? In the
latter case it could also be that the ligation mixture contains some toxic
component that kills the bacteria. You can check the ligation by running
the ligated material together with unligated material on an agarose gel.
Some time ago I had also problems with easy clonings. In my case, it turned
out to be the agarose. DNA that was isolated from this agarose (Kodak) with
Qiaex gel extraction was not clonable most likely because of a contaminant
that killed the competent bacteria. When I repeated the whole procedure
either with low melting agarose (isolate DNA by freeze/thawing) or with
normal agarose from another brand (Pronarose of Hispanagar; isolate DNA
with Qiaex gel extraction), I yielded normal numbers of colonies. Now I
have been using Pronarose in gel extraction experiments routinely without
any problems. I don't think that the Pronarose is special because of the
fact that these gel extraction kits are used quite succesfully in many labs
(I cannot imagine that they all use the same agarose brand).
>
>Good luck,
>
>Nico Dantuma 
>
>
>At 20:53 1998-08-08 +0200, you wrote:
>>In article <6pl4mh$bb1 at panix3.panix.com>, iayork at panix.com (Ian A. York)
wrote:
>>
>>>We're having a terrible time getting what should be a straightforward
>>>ligation to work.  I have a feeling we neglecting something obvious, so I
>>>won't be offended at simple suggestions ...
>>>etc. etc...........
>>
>>
>>Hello Ian !
>>
>>Just an idea: you may try to use a different bacterial strain for
>>transformation. I can remember a similar problem, that I experienced some
>>years ago. At that time it seemed to be just impossible for me to get a
>>1.5 kB insert into a bacterial vector ("pBacPak9", Clontech) designed for
>>constructing recombinant baculoviruses. I got plasmids with inserts, but
>>with the wrong size.
>>When I decided to change the bacterial strain (I had used E. coli XL
>>supercompetent bacteria from Stratagene) and to use E. coli DH5 alpha
>>competent cells, everything suddenly worked fine.
>>
>>Good luck!
>>
>>Thomas Burmeister
>>Free University Berlin
>>
>>
>>
>>
>>
------------------------------------------
Nico Dantuma
Microbiology and Tumor Biology Center
Karolinska Institute
Box 280
S-17177 Stockholm
Sweden
Phone: + 46 8 7286281
Fax: +46 8 331399 	



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