PCR subcloning woes :-(

John Nash nash at nrcbsa.bio.nrc.ca
Tue Nov 9 11:25:19 EST 1993


In article <931109080428.28810 at uabcvsr.cvsr.uab.edu>,
 <txpljfg at UABCVSR.CVSR.UAB.EDU> wrote:
>> This is a continuous problem and one in which you are not alone.
>> The problem seems to be nibbling caused by Sma and/or CIAP and the problem
>> can be minimized by careful titration of your enzymes - you will find that
>> you will need very little CIAP. Boehringer enzymes tend to give the lowest 
>> levels of background. Lately we have been so frustrated with this problem
>> that we have gone back to CsCl preps of our plasmid rather than Quiagen 
>> or Magic MiniPreps and we undercut rather than over-digest. A blue
>> background is better than a lot of false whites.
>> 
>> Incidentally, Amersham used to make Sma cut mp10 and this was superb.
>> They eventually stopped producing it because they too had quality control
>> problems - presumably they suffered the same difficulties.
>> 
>> Cheers from Down Under
>> 
>> Dave Saul
>>  
>> 
>Thank you very much for your note.  We sequenced a few of these white
>colonies a couple of days ago and found that there was indeed a
>frameshift in the lac gene at the smaI site: one to two base pairs (1-2
>G) had been removed from the cut site.  It is clear that this is a
>highly batch dependent problem.  I used this method with almost zero
>backround for over a year.  My problems may have begun when the batch
>of enzymes changed.  I am also considering that a partial source of the
>nuclease could be from the plasmid prep itself.  I do not routinely use
>CsCl preps, instead I use an alkaline lysis method.  Since I have not
>been routinely phenol extracting the preps, it is possible there is
>some contamination there as well.
>
>Regards,
>
>James F. George, Ph.D.
>University of Alabama at Birmingham
>205-934-4261 voice
>
>txpljfg at uabcvsr.cvsr.uab.edu

I always had grief when trying to clone blunt (incl. PCR-generated)
fragments into SmaI in the M13mp/pUC polylinkers, so I gave up on
using SmaI and switched to cutting the vector with HincII (which cuts
once in the polylinker at/around the SalI site), and have had few
problems... If I need to clone SmaI <--> SmaI, I use Xma I - which
gives staggered ends.


-- 
John Nash                           (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences,  National Research Council of Canada,
                 Yet another Aussie-in-exile ;-)
      *** Disclaimer:  All opinions are mine, not NRC's! ***



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