Long RT-PCR

[Brian Foley]

Jim Owens (jow at helix.nih.gov) wrote:

: I'm interested, too.  I have been performing RT-PCR on translocated c-myc
: loci in mice and have found repeatedly evidence that the cDNA synthesis
: has made longer products than I can PCR.  That is, I can amplify several
: segments less than a kilobase but cannot amplify the full 3-4 kilobase
: cDNA.

: Good luck,

: Jim Owens

	I found that if I used poly-T primer for reverse transcription
and I could PCR-amplify a small region near the 5' end of my mRNA (i.e.
far from the poly-A tail) but could not amplify the full 2.6 kb message
it indicated that my PCR reaction was failing, not the reverse transcription.
	I did all this before hearing about Wayne Barnes.  So I just worked
at optimizing the PCR reaction.  I did a 10-fold serial dillution of a 
plasmid in which I had cloned two small PCR products such that when using
the primers I needed to use to amplify the whole cDNA, I would amplify
the whole plasmid (minus a small region).  Thus I was able to find
a dillution of the plasmid that resulted in very littel PCR product.  I
then used this dillution to optimize the PCR conditions with the
very primers that I needed to use in the rt-PCR.  I found that if I used
more plasmid, it was impossible to find optimal conditions.  They
all worked fine.  In fact the conditions that give maximum yield of
PCR product when I use just a few copies of plasmid DNA give a poor
yield of PCR product if I use 100 nanograms of template DNA to start.

	Now that I know about Wayne Barnes and all the recent 
developments in PCR amplification of large fragments, I would
try some of the other tricks, such as using mixes of polymerases.
The bottom line is that it is quite a bit more dificult to amplify
fragments larger than 2 kb than it is to amplify fragments less
than 1 kb in length.  My optimum conditions included lengthening the
elongation step at 72 degrees to 3 minutes per cycle, and using
30 cycles.  1 minute elongation and 20 cycles was always good
for 300 to 600 bp fragments from the 5' or 3' end of the
same cDNA.

--
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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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