I have loaded my qPCR products onto a 2% agarose gel and ran them at 60V for 1 hour. I ran 2.5ng of cDNA in a 10uL qPCR reaction. As you can see in my image there is smearing of all bands in the heavy direction. I have tried reducing the amount of sample loaded and switching to TBE buffer and this helped slightly but still gives the smearing that is shown in the attached image. What else can cause this?
In our laboratory, we have the same problem, but with normal PCR. Even in NTCs there is a slight smearing, though no band appears in there even after 40 cycles amplification. We are working with Phire Hot Start II Polymerase from Thermo Scientific, using the attached 5x PCR-Buffer, the electrophoresis is done in 2.5% agarose gel with TBE buffer.
Two guys were now working for quite some time on this problem and they varied most parameters, without much success. Working with filter tips didn't improve the situation, neither did the reduction of primer / template concentration or the addition of DMSO.
The smear could be slightly reduced by increasing the amount of MgCl2 in the master mix from 1,5 mM to 3 mM. While this modification didn't solve the problem entirely, it reduced the smear to at least tolerable levels when using 25 cycles amplification.
@Artur Burzynski:
We will try out your suggestion tomorrow.
Your ladder migrates OK, so the problem is most likely in your samples. Run one of them through a cleanup column and see if the problem persists.
Another possibility is your gel loading buffer -are you using the same gel loading buffer for your ladder and your samples?
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