PCR subcloning woes :-(
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.
>James F. George, Ph.D.
>University of Alabama at Birmingham
>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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