RNA quantitation

at sunmail.lrz-muenchen.de at sunmail.lrz-muenchen.de
Mon Jun 17 10:37:40 EST 1996


R. Woodward wrote:
> 
> Dear All,
> I am looking for a reliable method for the quantitation of
> in vitro transcribed RNA. If its quick and cheap alls the
> better.

It depends a bit what you want to do with your RNA...For microinjection 
into Xenopus oocytes and for in vitro translation we just put the RNA on 
an normal (!) agarose-gel. You don´t have to work RNase-free, just do as 
following: prepare everything, put the prestained (ethidiumbromid) gel 
into the chamber, fill it up with buffer. Then quickly  mix your RNA 
with some sample buffer on the inner site of PARAFILM and load the gel. 
Run it for 10 minutes at 100V and take a look/picture. For those 
applications this is enough, you can see if there were RNAses (bands 
appear smeared) and with a lillte experience you will know if the amount 
is enough.
We also tried UV-densitometry but this is not as reliable as it could 
be.


> 
> Also does anyone else have problems with the Ambion capped RNA making
> kit, Ithink they call it the mMESSAGE mMACHINE. 

We use the T7 and the SP6 kit from Ambion (mMESSAGE mMACHINE) and we 
have very good results. It sound a bit you might have some contamination 
- check your pipette tips and so on. If it doesn´t help, add 1ul RNasin 
(Promega). It should work then..
Sandra
u7g11at at lrz.uni-muenchen.de

We seem t get such
> variable results in term of yield its untrue, even when we make
> RNA from the same sample of linearised DNA (But on different days)
> 
> Robert
> 
> R. Woodward
> 
> Email rw200 at cus.cam.ac.uk
> 
> d



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