No bands in PCR after DNase treatment
Posted 18 February 2013 - 03:21 PM
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.
Posted 19 February 2013 - 12:59 AM
Posted 19 February 2013 - 07:32 AM
Posted 19 February 2013 - 08:58 AM
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?
Posted 19 February 2013 - 09:53 AM