No bands in PCR after DNase treatment - (Feb/18/2013 )
Hi, I have been having some trouble with PCR lately. For my experiment, I must use PCR primers that are in the same exon, so I have been including negative controls to make sure I have no genomic DNA contaminations: a no template control, and cDNA reactions with no reverse transcriptase (-R.T.). I never get a band in the no-template control, but see bands in the -R.T. lanes that are the same size, although less bright. I figured this was genomic DNA contamination, so I treated the RNA with DNase prior to making cDNA. (I ran the RNA on a gel, too, and saw some extra bands other than the 18S and 28S bands). I did the same PCR, and when I ran it out on the gel... completely blank, no bands in anything (including my positive control, beta2microglobulin).
So my questions are:
1. Is there anything other than genomic DNA contamination that would produce dim bands of the same size?
2. Why would DNase treatment completely eliminate any bands? It says it is free of RNases.
something might have gone wrong in cDNA preparation? Because RNA is there that you confirmed on gel but where is the confirmation of cDNA? Faint band would be from genomic DNA contamination only and DNase treatment has not eliminated your RNA that is also sure with your gel.
Did you stop the DNase reaction before attempting to make your cDNA (some kits come with a specific DNase STOP solution)? What kit are you using?
neuron, I ran the RNA on a gel before treating with DNase, so one thing I can try is running it after treatment. The manual said there would be smears from the salt concentrations, but at least I can make sure something's there.
jerryshelly, I am using the Promega RQ1 kit, and yes it does come with a "Stop" solution which I used according to their protocol.
I am wondering if the Mg concentration in the DNase kit is interfering with the subsequent steps (either cDNA synthesis or PCR)... I treated 0.5 ug in 5 uL, used all of that for cDNA synthesis (20uL reaction), then used 2 uL of that for my PCR. If that's the problem, I can do a phenol-choloform extraction. (protocol also suggests diluting the DNase buffer or using an alternative buffer, i.e. the cDNA or PCR buffers). Any thoughts on if that could be the problem?
It sounds like everything you are doing is correct. The only other thing I can think of is your RNA is damaged in some way. How was the RNA prepared? Stored? How old is it? These can all affect the stability of your RNA. It is troubling that you ran your initial RNA and saw bright bands for both 18S and 28S. When you did this you saw prominent bands at the expected size or was the gel more of a smear product with faint bands throughout? Triple check your RNA by running it on gel with RNA you know has 18S and 28S in it. It may seem run-around, but it is the only advice I have.