genomic PCR help

John Ladasky jladasky at cmgm.Stanford.EDU
Tue Jan 13 21:43:01 EST 1998


In article <34BB9D2E.5072 at brown.edu>,
Jennifer Pan <Jennifer_Pan at brown.edu> wrote:
>Greetings, 
>
>I plan to do a ~10kb genomic PCR. Without any experience in the field, I
>wonder if there is a summary of dos and dont's , which enzyme has the
>best performance, Mg++ concentration, etc etc, or if you know these
>things?

Hello, Jennifer,

	I'm currently doing 5-6 kB PCR's from mammalian genomic DNA
using Stratagene's Taq Extender reagents.  Others on the newsgroup have
reported good results with other kits.  My recommendations have nothing
to do with enzymes or magnesium concentrations, but they greatly improved
my yields:

	1) Primers should be HPLC-purified.  If you use Taq Extender, the
	   penultimate three residues of each primer should be 3' phosphor-
	   othioate nucleotides rather than conventional nucleotides.  The
	   3' phosphorothioates block primer editing by Pfu polymerase.  I
	   don't know whether this modification helps with any other proof-
	   reading enzymes, but it might.  
	   (Skerra A, Nucl. Acids Res. 20:3551 [1992])

	2) Beware the "bad seed."  Something weird can happen in long PCR,
	   in which a contaminant enters a reagent, and then high molecu-
	   lar-weight smears are obtained rather than bands.  This has been
	   my most frequent long PCR problem.  If you don't see defined bands
	   from the beginning, even bands that you don't want, you probably
	   have the "bad seed."  Use filter tips, and periodically bleach
	   your pipet barrels.  Start with fresh reagents that haven't been
	   handled much when you are starting your procedure.  This includes
	   your genomic DNA.
	   (Hengen P, TIBS 19[8]:341 [1994])

	Good luck!

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



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