in vitro transcription question

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Aug 9 17:07:08 EST 1994


In Article <thompsop.1126295136L at Organpipe.UUG.Arizona.edu>,
thompsop at CCIT.arizona.edu (Paul Thompson) wrote:
>Hi, 
>I am trying to set up an in vitro transcription system, from following the
>literature I have found that some labs use a stop solution consisting of
>25mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% SDS and containing proteinase K and
>Yeast tRNA, to terminate the reaction. I do not have much expierence working
>with RNA, and RNAase free reagents, and my question is how do I pH a Tris
>buffer and still keep it RNAase free? Tris reacts with DEPC I believe, is it
>therefore feasible to use DEPC treated water in making up this buffer? 
>Thanks in advance for any suggestions.
>Paul T.  

In my experience, it is not necessary to STOP the reaction. If anyone else
knows of a reason, I would like to hear it. To answer your next question, I
usually adjust the pH of the tris, then remove an aliquot to test it with a
pH meter. I guess you could use pH paper if you have one you like (trust?)
and are making a small volume of buffer. Third, it is true, DEPC is not an
effective means of removing RNAses from tris. What I have done in the past
is, get a new bottle of tris, wear gloves when you open it, and use it only
for making up RNAse-free solutions. I have also used sterile, disposable
spatulas or spoons to get the tris out of the bottle. You can use
DEPC-treated water to make the tris solution, but make sure you autoclave
the water after treatment, as some DEPC residue will still be there.
Tracy Aquilla, Ph.D.
Dept. of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aquilla at salus.med.uvm.edu



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