8 replies to this topic

[Ikar]

Hi,

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!

Best regards,
Ikar

[hobglobin]

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?

Edited by hobglobin, 05 February 2012 - 09:41 AM.

[Ikar]

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.

[phage434]

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.

[Ikar]

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...

Edited by Ikar, 05 February 2012 - 11:30 AM.

[phage434]

Put it in a screw top vial and add it to boiling water for a half hour.

[Ikar]

Is it really necessary to boil my dna extractions for 30 min or is sth. like 10 minutes not sufficient?

[phage434]

No, it is only necessary to boil the RNAse once.

[mdfenko]

what ikar is missing is that you boil the rnase solution before using it with the dna prep.