a good PCR mimic?
bgrobben at uia.ua.ac.be
Fri Jun 21 10:46:57 EST 1996
I'm currently trying to optimize a quantitative RT pcr for a certain
mRNA in different rat tissues. In order to make a good quantification, I
needed a nice internal control. I first tried to create a deletion
mutant of the cDNA I'm working with, thereby reducing the length of the
PCR fragment from about 800 to 650 bp.
When doing PCR over 40 cycles on strong dilutions of plasmids containing
the full length cDNA, and shorter MIMIC cDNA, I always am confronted
with an intermediate PCR product, probably a heterodimer of a long and
short fragment. I tried to circumvent this by raising primer
concentration (I used 16 pmoles of each primer per 20ul reaction) and
lowering the plasmid concentration. But the intermediate stayed, while I
got a clear primer-band on my gel...
Probably it is better to synthesize another PCR-mimic, with different
sequence. I thought of doing this by annealing both primers to a
restriction digest fragment of a nice lenght (about 500 bp) with 5'
overhangs, by means of T4 RNA ligase (we have this available in the
lab), that anneals ssDNA or RNA. Afterwards, this product should be
blunted, cloned, and a selection must be made to select for fragments
containing different primers at the ends.
Has anyone experience with this technique, and does anybody knows
whether T4 RNA ligase can be used to ligate primers to blunt end
fragments also. I know there are more easy ways to create such a
fragment, by doing PCR with primers containing both
'stuffer-DNA-primers' and primers for use in the quantitative PCR, but
that looks more expensive, since we have all the material for the T4
ligase method at our disposal...
PS: can anyone tell me how to subscribe to this mailing list (I suppose
this is a mailing list) since I wrote this article by WWW-ing to the
archives of this list on the net.bio.net-site....
Lab. Cellular Biochemistry
University of Antwerp
email: nicolai at uia.ua.ac.be
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