Strange large bands on my PCR gel, has anyone seen this before? - PCR (May/14/2008 )
Im getting strange genomic sized bands (gel 1), any ideas on what is causing this and is it related to the failed reactions? I am trying to amplify the ras-related protein gene of Phytopthora(~400bp). I have tried ethanol precipitation and diluting the DNA (1:1, 1:5 &1:10) and the large bands are still there!! Any help would be greatly appreciated as it is difficult to get more cultures for these samples plus it is driving me to the bad karma hypothesis!!! I have also attached the DNA gel (gel 2).
Are you amplifying cDNA? If so, could it possible you have genomic contamination? Did you use DNAse?
You could digest the PCR reaction with DpnI before you load the gel. All genomic DNA gets degraded, but the PCR product doesn't.
Funny how a couple of the worst lanes (Gel 1, lanes 6, 7, 13, 14) are missing one of the primer band/smears. What PCR conditions are you using?
Also, how did you prepare the template DNA?
Matt, you need to give more details, but on the face of it, these are clearly genomic DNA bands. If it is RT-PCR, you have genomic DNA contamination, and the PCR has failed. If it is genomic PCR, everything is fine, except that the PCR failed in some cases, and worked in others. In that case, I would suggest purifying genomic DNAs again and repeating the PCR.
May be you need to design more robust primers and PCR conditions: in my experience, 400bp PCR generally works, even if the DNA purity is not that great.