Extraction of gram positive rRNA without a centrifuge

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Jan 18 07:52:26 EST 2001


In article <945j75$3lr$1 at nnrp1.deja.com>, the eminent  at Deja.com wrote

Can't really answer on best method to extract RNA from gram positives
but a search through Biotechniques will turn up umpteen protocols over
the last few years.

>Does lysozyme destroy RNA?

Only if it has RNases in it.

>Does mercaptoethanol effect the activity of lysozyme?

Pass

>Does heating and cooling destroy RNA?

It all depends on what you call heating and in the presence of what.
Heating in the presence of Mg is to be avoided at temps above say 65C
for any length of time.

>What is a good anti-inhibitor agent to use in RT-PCR (BSA seems to be no
>help?

Anti-inhibitor for what? RNAses? BSA will have no effect on RNAses.
There are various RNase A inhibitors but they will have no effect on any
other RNase.

You could always harvest your cells and lyse by putting straight into
Trizol. I know of a group that always added DEPC to their cells prior to
lysis in a non-Tris buffer. Seemed to work well.

Duncan 
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk






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