RNase protections assays. Too much salt?

Jim jmeador at adnc.com
Mon Apr 1 10:09:09 EST 1996


In article <4jeoah$1kt6 at hearst.cac.psu.edu>, bms8 at psu.edu (Brian M.
Shewchuk) wrote:

Daniel Pereira <mgs6145 at student.health.ufl.edu> wrote:

>Angela Hofstra <Hofstra at aa.wl.com> wrote:
>>
>> Hi,
>> 
>> Lately we've been having trouble with the last precipitation step in our 
>> RNase protection assays. We follow the procedure essentially as outlined 
>> in "Current Protocols in Molecular Biology".  After precipitating in 
>> ethanol we get lots of white precipitate, which I suspect is largely 
>> salt.  When we dissolve the pellet in loading buffer and load it on a 
>> vertical gel the sample rises.  
>> 
>> Has anyone encountered this before and what can we do about it?
>> 
>> As well I'd like to get people's opinions of the Maxiscript and HYBSpeed 
>> RPA kits from Ambion.
>> 
>> Thanks,
>> 
>> Angela 
>> 
>> 
>> 
>If you think it salt- I would wash the pellet in 70% Ethanol- then load
>As for Ambions HYspeed kit- I have had good sucess with it. 

I use the same protocol and get the same ppt.  It's all the NaCl in
the RNase digestion buffer.  So, 70% EtOH doesn't solubilize it (I've
tried several washes, with no decrease in the ppt.).  It dissolves in
the formamide-based loading dye, but then all the salt creates a
massive electrophoretic blockage then you run the gel.  It takes close
to an hour for the mass of ions to run out before the samples can even
enter the gel, and if you don't let it run long enough,  the positions
of your protected fgmts. will be anomalously high on the gel.

Let me know if you find a solution.

Brian
The Department of Biochemistry and Molecular Biology
The Pennsylvania State University


Brian,

The obvious solution is to reduce the salt. There does not need to be much
salt in the reaction anyway. Also you can use NaAc which I think is better
with 70% EtOH. The salt is there merely to increase the hybridization of
the duplex RNA, but it actually decreases the activity of most RNAses.
Since you can increase the hybridization of the duplex via temperature as
well as salt, there should be no problem with reducing the salt conc.


Hope this helps,
Jim



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