Good Rnase free DNase sources?
pearton at saul6.u.washington.edu
Mon Oct 27 13:34:09 EST 1997
brett (brett at BORCIM.WUSTL.EDU) wrote:
: >S. MORI (smori at NMSU.Edu) wrote:
: >: Dear molbio's
: >: I am trying to get a hold of some good RNase free DNase... I had some 1
: >: year old Stratagene stuff, but the freag'n thing ate all my RNA... It had
: >: been stored at -20C... It may have been the age (I doubt it) but I don't
: >: wanna risk it again.... anybody has know of a better source?
: >I've always had good results with the DNase I from GibcoBRL - the RNA prep
: >is immediately usable in RT-PCR, and I assume, is good enough for
: >northerns (although I don't usually bother for northerns).
: >Dave Pearton
: >pearton at u.washington.edu
: Dave, have you ever shown that your RT-PCR is free from DNA by doing the RT-
: control? While we find that DNAse I and RQ1 DNase do indeed destroy DNA, it
: is not nearly quantitative. That is, =>0.1% of DNA remains by qPCR (RT-).
: Someone in our lab has been trying various combinations of nucleases and is
: now able to decrease the DNA titer to undetectable levels, but not with only a
: single step. I suppose then it depends also on the sensitivity of your specific
: PCR (ours can detect 1 molecule DNA ~30% of the time). I'd be glad to hear your
Yes, the -RT pcr control is clean in all the primers I've used (which is a
fair number). I've also used intron-spanning primers and only found the
processed cDNA product. Of course my primers might not be as sensitive -
I've not tested the limits of detection, but I'll be happy to try your
primers if you like.
pearton at u.washington.edu
Down he sank in a chair-ran his hands through his hair- +
And chanted in mimsiest tones +
Words whose utter inanity proved his insanity, +
While he rattled a couple of bones. +
"The Hunting of the Snark" Lewis Carroll +
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