DNase treatment of RNA sample

Virginia Dress Dress at biosci.arizona.edu
Tue Mar 14 17:50:41 EST 1995


In article <199503141158.UAA02322 at bekkoame.bekkoame.or.jp>,
tishizuk at AQUARIUS.BEKKOAME.OR.JP (Toshiharu Ishizuka) wrote:
> 
> Dear bionetters
> 
>         I would like to thank all people who responded my problem. Most
> people recommended me the pilot experiments to determine which component
> contains RNase. I did the experiments and found that the buffer for DNase
> treatment contains RNase. Thus, I would like to ask you. 
>         How can I prepare Tris-based DNase buffer without RNase ? I heard
> that diethylpyrocarbonate reacts with Tris, and it is difficult to
> inactivate RNase by autoclaving. 
> 
> The composition of our DNase I buffer is 
> 10mM Tris-Cl, pH8.3
> 50mM KCl
> 1.5mM MgCl2
> 10U human placental RNase inhibitor (TOYOBO)
> 10U RNase-free DNase I
> 
> I appreciate if anybody teach me.
> 
> Sincerely yours,
> 
> Dr. Toshiharu Ishizuka
> Department of Biochemistry, Chiba University, School of Medicine
> 1-8-1, Inohana, Chuo-ku, Chiba, 260 JAPAN
> FAX +81-43-226-2041
> E-mail
> QFH03407 at niftyserve.or.jp
> tishizuk at aquarius.bekkoame.or.jp

So the important things I've read are:
					1)  Have a separate bottle of reagents for RNA work only (i.e. keep
them
												RNase free).  Especially things like Tris and EDTA, but if you
can
												afford it, do it for the salts too.  It's up to you whether or
not
												you want to buy the molecular biology grade guaranteed RNase,
DNase
            free reagents, I don't know if they are actually cleaner or
just
            tested and certified.
					2)  Always handle the reagents with RNase-free articles:
												a)  Always wear gloves
            b)  Use baked spatulas and glassware for weighing out and
making
																			the solutions or use disposable plasticware for
solutions.
            c)  Use RNase free water - either DEPC-treated (DEPC
inactivated)
                    or test your local DDW source, it could be RNase free. 
I've
                    read that most Millepore double-deionized water is
RNase
                    free.
				I rely a lot on disposable plasticware to eliminate a lot of RNase
problems,
any bag of microfuge or larger tubes, pipet tips, etc. which have not been 
touched with bare hands is generally considered RNase free.  I've also
heard it
is better to use them as is rather than autoclaving them to sterilize.

			I think I've probably forgotten something(s), but that's a start and I'm
sure
someone else will fill in the omissions.

Good luck,
       Ginnie



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