High RNase concentration interfering with PCR?
Posted 05 February 2012 - 09:00 AM
I started working in a new lab last week and their protocols seem a little bit awkward to me.
Referring to a DNA extraction protocol from cherry fruit flies:
After they extracted and precipitated the DNA and dried it in the SpeedVac, they add TE buffer + RNase and let it dissolve over night at room temperature. I was told to add RNase A to an end concentration of 10mg/ml in the buffer. In my opinion this seems to be a little bit to high? I am afraid that it could interfere with the PCR reaction.
In fact the PCR reactions did not work, I used older DNA sample they gave me and samples I isolated by myself. However, I used a lambda test kit in parallel, which worked perfectly fine. So I am thinking that either the primers (although I tried several ones) might be to old (they told me that they successfully used the same primers in the past with the same PCR program) or there is really a problem with an too high concentrated RNase.
I would really appreciate it if you have some ideas for me!
Posted 05 February 2012 - 09:18 AM
Are you sure that the RNase is free of DNAse?
Edited by hobglobin, 05 February 2012 - 09:41 AM.
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that did belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
Posted 05 February 2012 - 09:49 AM
Posted 05 February 2012 - 11:16 AM
Posted 05 February 2012 - 11:28 AM
Edit: I might also want to check if the RNase stock contains DNase first...
Edited by Ikar, 05 February 2012 - 11:30 AM.
Posted 05 February 2012 - 01:31 PM
Posted 07 February 2012 - 01:02 PM
Posted 14 February 2012 - 11:12 AM
talent does what it can
genius does what it must
i used to do what i got paid to do