questions about RNase A storage - enzyme manipulation (Aug/10/2005 )
I am working on RNA stability against RNase A digestion. I dissolved RNase A (powder, Roche) in [10 mM Tris, pH 7; 5 mM CaCl2, 50% glycerol].
Question 1: Is that the correct way to dissolve and store RNase A?
Question 2: Should the Rnase A solution be stored under -20 C or 4 C is ok?
Question 3: I figured out a concentration of RNase A which can cleave a 80-mer RNA within 30 min. But when the same concentration of RNase A was used with a 21-nt RNA (at the same amount as the 80-mer RNA), no digestion was observed throughout the 40-min incubation. Does anyone have similar observation, or is there something wrong?
Any suggestion or idea would be really appreciated! Thanks in advance!
resuspension of RNAse A depends on the manufacturer. I've once followed the maniatis rotocol and get the protein precipitated. The manual of manufacturer told RNAse should be dissolve in acidic TE and the pH ajusted... So see the manufacturer's advice.
For storage, i've made aliquots of RNAse. I use one stored at 4° for mini/midi/maxipreps and it's ok.
RNase A only cleaves RNA between certain bases - I think purines but I am not certain You may not have the right sequence in your RNA oligo to be cleaved by RNase A and you might need to use another RNases (say RNase T1).
DNA sequencing analysis software