Variable PCR results - Ideas and Suggestions?

Doug Jones jonesd at telenet.net
Tue Oct 21 08:35:03 EST 1997


I have been having a problem with an RT-PCR for murine IL-6.  After
optimization, which went well, I have been unable to obtain consistent
results, even in the same run.  

The PCR is done in a mix using 1.5 mM MgCl, 50mM KCl, 10mM Tris, 0.1%
Triton X-100, .1% NP-40, .1% Gelatin, 0.25 mM each dNTP's, 5 U Taq
(Promega) and primers in a volume of 50 microliters.  The cycle is 1
min at 95, 1 minute at 55, and 1.5 minutes at 72 degrees, for 35
cycles in a Stratagene RoboCycler.  

The problem is that there is run-to-run inconsistency, with bands
either appearing or not in the spleen cDNA, and within run
inconsistency - I have run 4 samples using the same mix, and the same
cDNA on the same run - and 1 shows a very nice band, 1 shows a fuzzy,
indeterminate band, and the other two show nothing.  

I am getting a bit desperate, trying to get this to work, so I'd
really appreciate any help!  Thank you.

Douglas Jones
jonesd at telenet.net 
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