LA-PCR problems

John Ladasky jladasky at cmgm.Stanford.EDU
Wed Jan 14 21:12:11 EST 1998


In article <4D8B01173D79D011BBD200805FEACC670219CD at LAUIMLS05.qc.dfo.ca>,
Parent, Eric <ParentE at dfo-mpo.gc.ca> wrote:
>I'm trying to amplify a 16Kb mtDNA, and always get a large smear from
>the well.  My negavive control dont show anything. Still, could it be
>"bad seed"(even with my control being ok), or just that my conditions
>are not optimise ??
>
>Thanks for your help

Hello, Eric,

	What is the range of molecular weights in the smear?  What is the
predicted annealing temperature of your primers, using a program like Oligo?

	If the smear is mostly above, say, 3kB, and you are annealing below
the computed annealing temperature of your primers by more than about five
degrees, I would definitely suspect the "bad seed."  Even more so because
mitochondrial DNA is present at a high copy number -- it should be much
easier to amplify than single-copy nuclear genes.

	During long PCR I've acquired the "bad seed" problem no less than
three times.  Two of those times, the problem affected all of my templates
and was solved by ordering fresh Taq Extender enzyme.  In the third situa-
tion, the smearing was confined to a single sample of genomic DNA, and by
preparing that DNA fresh from the cell line, I corrected the problem.  Is
the genomic DNA sample that you are using one that you have handled a lot?  
Can you try other samples?  If you get the same smear with all of your 
templates, its probable that the "bad seed" is in one of your reagents.

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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