Urgent request: BME required for RNA isolation?
dbell at fresno.ars.usda.gov
Mon Feb 7 12:54:18 EST 2005
I would definitely add the BME as soon as possible.
BME maintains a reduced environment preventing oxidation. You probably
nitice a lot of browning in your pellets. You may be able to clean up
your prep with a few alcohol precipitations. But your RNA is probably
You should try an RT-PCR with 'good' RNA and compare it to your RNA.
But my gut instinct is to tell you to do those 60 samples over again
with the BME.
From: owner-methods at hgmp.mrc.ac.uk [mailto:owner-methods at hgmp.mrc.ac.uk]
On Behalf Of atari1980 at hotmail.com
Sent: Monday, February 07, 2005 9:15 AM
To: methods at hgmp.mrc.ac.uk
Subject: Urgent request: BME required for RNA isolation?
OK, I screwed up. We use the Qiagen RNeasy kits to extract our RNA from
tissues. With the last kit, I forgot to add the beta-mercaptoethanol to
the lysis buffer (ahrrrgg!). I had homogenized almost 60 samples with
this lysis buffer before I relized my error.
Here's my problem - when I read the final RNA extracts on the spec, the
260/280 ratios were right around 1.9-2 and I the amount of RNA extracted
(based on the 260 reading) is about the same as my previous extractions
with lysis buffer containing BME.
Does anyone know if the RNA extracted without BME is still adequate for
RT-PCR? Any suggestions would be greatly appreciated. I still have
about 80 more samples to extract, and now I am reluctant to change my
protocol midway though by adding the BME now.
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