RNase A in Qiagen buffer P1
 

Hi all I have a question about RNase A and column vs non column minpreps.

In Qiagen RNase A is added to the initial resuspension buffer. I am unsure
as to what good this does as in P1 the RNA/DNA is still in the bacterial
cells. When the alkaline lysis buffer is added in P2 the DNA/RNA is
released but I read RNase A is inactivated in .1% SDS (P2 is 1% SDS). In
any case RNA generally does not bind to the silica columns that much anyways
so the columns remove the RNA.

I have been using the Qiagen buffer to do the alkaline lysis then doing
phenol/chloroform extraction followed by ethanol precip. When I do this at
the end there is a bright RNA band on the gel around 50-100bp. Qiagen
claims in their manual this is RNase A-resistant RNA.

My question is will precipitating the DNA/RNA after the lysis and
resuspending in TE/RNase then doing PC extraction and another alcohol
precipitation remove the RNA because RNase A is inactivated in SDS or is
Qiagen correct in saying this RNA is simply resistant?

RNase A in Qiagen buffer P1
 

"S. Schoenfeld" <sschoe2[remove this spam tag]@uic.edu> wrote in message
news:cqf32j$dtt$[Only registered and activated users can see links. Click Here To Register...].uic.edu...

It's easily tested!

But I'm guessing it should work. After all I do crude minipreps in
essentially that way, and I have no RNA to speak of.

As to what good does the RNAse in P1 buffer... well, all I can say is that
the DNA you recover afterwards has no visible signs of RNA, so it *must*
work.

Jose

Re: RNase A in Qiagen buffer P1
 

Well the RNAse has a half life of 6 minutes. The second step doesn't last nearly that long so the RNAse is not deactivated before the RNA is cleaved. So despite the high concentration of SDS, the RNA is still cleaved.