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Preparation of RNase A From Powder


Best Answer bob1, 07 December 2013 - 12:38 PM

RNases are incredibly stable - they will denature at 100 C, but will then re-fold and start working again.  This is one of the reasons people think that RNA based organisms preceded DNA based.

 

RNase is fine in pretty much any solution, I usually make mine up in water.

 

RNase shouldn't interfere with the restriction enzymes, but it would be much better to clean up the DNA before digesting as REs can do funny things and are easily inhibited by steric hindrance, so any RNase bound to the DNA (yes, they can partially digest DNA as well - make sure your incubation conditions are correct), could potentially stop the DNA from being fully digested.

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#1 djvan

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Posted 07 December 2013 - 10:55 AM

I recently ordered some RNase A from Roche that came in a dried powder form.  I went online to get instructions on how to Prepare RNAse A, only to find out that this product has no instructions!

 

Sigma has a package insert online for their RNase A.  They suggest:

 

1) Preparing a 10mg/mL solution in 10mM sodium acetate, pH 5.2

2) Heat to 100 C for 15 minutes.

3) Allow to come to room temperature.

4) Add .1 Volume of 1 M Tris-HCl, pH 7.4

5) Aliquot and store at -20 C

 

This is how I decided to prepare my RNase A.  I later read that RNase A can withstand temperatures up to 100 C - sure hope heating it at 100 C didn't denature it!

 

Now to remove RNA from a DNA solution (already isolated using a kit):

1) Added stock RNase A to a working concentration of 10 ug/mL - my sample was in 1x TE.

2) Incubated at 50 C for 20 minutes.

3) allowed to come to room temperature

4) Stored at 4 C.

 

Today I'm going to restrict my DNA sample - will RNase A affect the restriction enzymes?  I plan to do a chloroform/phenol extraction afterward..

 

Thanks!



#2 bob1

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Posted 07 December 2013 - 12:38 PM   Best Answer

RNases are incredibly stable - they will denature at 100 C, but will then re-fold and start working again.  This is one of the reasons people think that RNA based organisms preceded DNA based.

 

RNase is fine in pretty much any solution, I usually make mine up in water.

 

RNase shouldn't interfere with the restriction enzymes, but it would be much better to clean up the DNA before digesting as REs can do funny things and are easily inhibited by steric hindrance, so any RNase bound to the DNA (yes, they can partially digest DNA as well - make sure your incubation conditions are correct), could potentially stop the DNA from being fully digested.






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