Variable PCR results - Ideas and Suggestions?
adobrovic at medicine.adelaide.edu.au
Tue Oct 21 20:05:46 EST 1997
I find that 5 Units of Taq is excessive and may even be inhibitory. We use
0.5-1.0 units depending on the application.
You might also try either decreasing the dNTPs to 0.2mM each or increasing
the MgCl2 to 2.0mM.
>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.
>jonesd at telenet.net
>(remove NOSPAM to reply from USENET)
Alexander Dobrovic, Ph.D.
Department of Haematology-Oncology
The Queen Elizabeth Hospital
Woodville, SA 5011, Australia
Affiliate Senior Lecturer
Department of Medicine
University of Adelaide
Tel 61-8-8222 6884
Fax 61-8-8222 6046
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