Variable PCR results - Ideas and Suggestions?
Tung Tran Duc Tuan
plg-ttdt at risoe.dk
Tue Oct 28 09:22:01 EST 1997
In article <344cac3d.2019757 at news.telenet.net>, jonesd at telenet.net says...
>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)
I have some suggestions:
Add about 5 % DMSO to the reaction mix to obtain an higher stringency.
Reduces the amount of Taq-polymerase to 1 U per reaction
Increases the concentration of MgCl2 to 2mM
Increases the extension steps duration to 2 min.
Use Touch-down PCR:
Use an relatively high annaeling temperature for the 6 firsts cycles, then
lowering this temperature with succesive cycles to en annealing temperature of
55 C over 15 cycles and keep this temperature for the reste of cycles.
More information about the Methods