PCR of a long genomic fragment... HELP?

Basavaraju Shankarappa bsh at MED.PITT.EDU
Thu Mar 18 19:02:06 EST 1993


Following is a repost of what Vivian had posted.  I hope it will be of good
help.  
	One of the following publications is very unique in that they 
have used Tricine instead of Tris.  The logic is that the pH of tris varies
or is not what you intend it to be at higher temperatures.  This fact is
overcome by the use of Tricine.  And they show that it is possible to amplify
6 kb piece from a plasmid in 10 cycles ( I cannot find the publication
so I feel bad for not giving the due credit!!).  The whole logic seems
very good.  
	Now my question is will this feature also improve sensitivity in 
routine PCR.  Has anyone done PCR using tricine on a regular basis for
the detection of very very low copy template in a genomic background 
similar to the detection of infectious organisms?  It seems like it
should help.  Following references are a good reference material for
anyone doing PCR.  Thanks to Vivian.
Raj Shankarappa
bsh at med.pitt.edu
In article <1mcfo5INNb0l at shelley.u.washington.edu>,
miza at hardy.u.washington.edu (Miguel Zamora) wrote:
> I'm having problems amplifying a 5.3 kb DNA fragmente using PCR. I've tried
> Taq Polymerase and Hot Tub (Amersham), and I didn't get anything. I know
> the reagents are OK because I get the right size product if I use two
> primers that are closer to each other. I've amplified some large fragments
> before (up to 2.6 kb) with no problem. Anyone has any experience in long
> PCR amplifications. Any suggestions? 
Have you seen (for starters!):

Schwarz et al, 1990 NAR 18 p1079 ("Improved yields of long PCR products
gene 32 protein")

Kainz et al,1992 Analytical Biochemistry 202 p46-49 ("In vitro
amplification
of DNA fragments >10 kb) ?

Ponce and Micol, 1992 NAR 20 p623 (they try a diff. buffer)

 I think when things don't work, (after you've considered the usual
 problem sources) frequently the problem lies in the oligos themselves.
Do you know that your two primers (for the desired product) will
work with each other?  You could try an internally deleted version of your
intended target (to separate out the effect of size) as template, if such a
thing
 is available.  Or at least try each oligo with some other primer that you
know
 works.   Could you possibly need to use a longer time for elongation? 
I've
 made a 7 kb piece with Taq via in a 2-step program that calls for
 1 min denaturation at 92C, then 8 min at 65C (for both annealing and
elongation).  I'm not sure that 8 min was crucial, but I'm just
passing this experience along, since it was part of my setup that worked,
 and may have helped.   Good luck!  :-)

From: vmiao at oregon.uoregon.edu (Vivian Miao)




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