Making cDNA for RT-PCR of N-terminal fragment

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Sat Apr 25 13:30:19 EST 1998


Hi folks --
    Several questions.  First, everyone "knows" that it is easier to PCR
up the 3' end of a cDNA transcript rather than the 5' end (when using
oligo dT for the cDNA), but has anyone published any evidence for this?
    Next.  The region I want to PCR from is 3.4 kb from the poly-A
tail.  What I already know is that Murine Rev. Transcriptase can be run
at 42'C and ought to be able to copy sequence that long (assuming my RNA
is intact).  I also know that I could use hexamers instead of oligo-dT
as my primer for cDNA.
    Thus far my luck with the murine RT is so-so.  I can amplify my
gene, but I want to do quantitative PCR on N-terminal variants of my
gene, and for quantitation, I do not trust the RT and my RNA to be good
enough; I can see it, but only after tons of cycles, and there are
dimers by then.  In terms of using hexamers, none of us here ever uses
hexamers, so...
    When doing quantitative PCR (or any PCR in a two stage process,
making cDNA first and then doing the PCR with aliquots), if I use
hexamers, do I poly-A select first?  The rule of thumb with quantitative
PCR was not to poly-A select (adds another variable), but rather to make
cDNA from total RNA.  How much hexamer do I use, how much RNA?
    Finally, how well does a cDNA reaction scale up (right now I use 2
ug total RNA, 25 ul H2O, 5 ul buffer 10X MuMLV (stratagene), 5 ul dNTPs,
3ul oligo dT (100 ng/ul stock), 2 ul Rnasin, Add RNA (in 10 ul) to rxn,
Add 1 ul MuMLV enzyme (50 U)).  I want to make 10X as much.  Is the
scale up (10X as much of everything) going to work?
    Thanks,
        Harry


--
Harry J. Witchel, Ph.D.
Dept. Physiology
Medical School
Bristol  BS8 1TD
   England

Harry.Witchel at Bristol.ac.uk
http://www.bris.ac.uk/Depts/Physiology/Staff/hw.htm





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