in vitro transcription question

Virginia Dress Dress at biosci.arizona.edu
Wed Aug 3 15:24:33 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.

Hi Paul,
					What I do is:
											1)  Assume that the 10N NaOH or HCl stock I use is RNase free, I
just                                         make it up in DDW, no DEPC, no
autoclaving, no filtering.
                 This is probably a pretty good assumption
											2)  I pH small samples of my buffer stock and throw them away,
so
																	that I never add anything back to the stock.  I use a pH
meter.
																	Others I know use pH papers which vary in reliability (
some
																	are good, some suck) but you could use those to at least
get
	                you into the ballpark region for the pH you want.
											3)  Make stocks of Tris base and HCl or Tris-HCl and follow one
of 
																	the tables on what ratio to mix them in to get the pH you
want.

Good luck,
          Ginnie
  



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