High RNase concentration interfering with PCR? - (Feb/05/2012 )
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!
For me it sounds too high too, though I'm not sure if a high RNase concentration intereferes. Perhaps the stock solution might have 10 mg/ml and working solutions later 10 micrograms/ml (as example; I used it and had no problems with PCR).
Are you sure that the RNase is free of DNAse?
Your suggestion also sounds more reasonable to me! I am not sure if it is DNase free, I hope so at least. I quantified the DNA after the extraction via NanoDrop and the concentration was quite good. 0.3 mg/ml. And the 260/280 was arround 2.
The quantification tells you nothing -- chewed up DNA from DNAse activity will be reported just as easily as intact DNA. You can inactivate DNAse in an RNAse preparation by warming to boiling for a while.
Ok, sounds convincing. What temperature would you recommend after adding the RNase, something around 90-100°C? And for how long??
Edit: I might also want to check if the RNase stock contains DNase first...
Put it in a screw top vial and add it to boiling water for a half hour.
Is it really necessary to boil my dna extractions for 30 min or is sth. like 10 minutes not sufficient?
No, it is only necessary to boil the RNAse once.
what ikar is missing is that you boil the rnase solution before using it with the dna prep.