RT-PCR problem.....HELP!

Simon Dawson mbxspd at unicorn.nott.ac.uk
Tue Jun 14 04:54:36 EST 1994


Hi there,
        I'm currently experiencing problems with some RT-PCR reactions.
The reactions themselves are working fine, it's the analysis thats
bombing.
  Currently, I reverse transcribe a mixture of total RNA (from
experimental samples) and in vitro control RNA. The control RNA is made
from a deletion construct of the cDNA I'm trying to assay. The deletion
in the control RNA is such that both the test and control RNA's can use
the same pair of primers in the PCR step.
  The problem is arising during analysis of the PCR products on a
standard, ethidium bromide stained agarose gel. What seems to be the
problem is that I'm experiencing some sort of heteroduplex formation
between the PCR products generated from the control and sample RNA's
(this problem dosn't occur if I leave the control RNA out of the
procedure)! That's making things messy.
  The PCR cycle used is:
  1) 96¡C for 3 minutes
  2) 55¡C for 30 seconds
  3) 72¡C for 2 minutes
  4) 96¡C for 15 seconds

  Cycle between steps 4) and 2) for 31 cycles in total.

  5) 55¡C for 30 seconds
  6) 72¡C for 2 minutes
  7) 4¡C for as long as it takes me to get the agarose gel set up!

  Now, unless I'm mistaken, surely all of the products should be
homoduplexes at the end of this program?
 Anyhow, does anyone out there have any suggestions as to a reliable
way of getting around this problem? Has anyone experienced anything
similar? Is it possible that my primer concentrations are falling too
low towards the end of the reaction sequence (I currently use ~20
picomoles of each primer per reaction, 200µM dNTP's).
  Any help/email's would be greatly appreciated!

                 Frustrated of Nottingham :)

Simon Dawson              Internet email: mbxspd at unicorn.nott.ac.uk
Dept. Biochemistry
Nottingham University
U.K.



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