HELP: Long PCR problem (urgent)

ladasky at my-deja.com ladasky at my-deja.com
Wed Mar 29 20:21:11 EST 2000


In article <E951D7A047E6D311BC2C00805FEACC6703FC62 at lauimls05.qc.dfo.ca>,
ParentE at dfo-mpo.gc.ca ("Parent, Eric") wrote:
> Dear Netters
>
> I'm trying to do PCR amplification of total mtDNA (16kb) of
> crustaceans and I'm getting frustrated. I read Barnes' paper in Proc.
> Natl (1994) about PCR amplification of up to 35-kb DNA. In that paper
> he refers to reactions that failed that he couldn't explain. That
> failure mode gave rise to massive ethidium bromide staining in the
> sample well, which is exactly my situation. I was wondering if anybody
> could help me explain those failed reactions and
> that staining in the wells?

Hi, Eric,

We had this same conversation, here in this newsgroup, about two years
ago.

http://x21.deja.com/[ST_rn=ps]/getdoc.xp?AN=298828424&CONTEXT=
954378204.962265088&hitnum=14

At that time, you were getting high molecular-weight smears instead of
specific PCR products.  I directed you to Wayne Barnes' follow-up
technical notes on long PCR -- Trends in Biochem. Sci. 19(8):341-342
[Aug. 1994].  I am grateful to Paul Hengen who, a year or so before our
exchange, directed me to the same article here in this newsgroup.

Barnes describes problems with an unknown contaminant that he calls the
"bad seed."  To eliminate the bad seed, discard all of your reagents and
replace them with fresh ones.  You can try replacing your reagents one by
one, but it isn't worth the hassle.  Always use filter tips, and
periodically bleach your pipet barrels.

I've found that I can never be careful enough when I'm handling genomic
DNA.  I keep master gDNA stocks from which I dispense aliquots.  As soon
as I get the bad seed problem, I discard the aliquot and get a new one.

Have you ever succeeded in obtaining a successful 16 kB amplificiation?
Is this staining in the wells the same problem that you had before, or is
it a fundamentally new problem?

Let us know!

--
John J. Ladasky Jr., Ph.D.
Department of Structural Biology
Stanford University Medical Center
Stanford, CA 94305


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