Good morning, I am trying to degrade an RNA naturally resistant to degradation by RNases, I wanted to use MNase but it only degraded very little. My sample is in liquid medium, in 95uL of RPMI and I added 5uL of Reaction Buffer for a final concentration of 50mM Tris•HCl pH 8, 5mM CaCl2 and heated at 37 degrees for 30 min in the water bath, then I also tried it for an hour and no good results either. I have used 0.5uL (50 Units) and 1uL of enzyme.
I don't know if I'm doing it right, could you please help me, I don't know anyone who works with this enzyme, I've also tried RNAse A/T1 and the RNA doesn't degrade either. Thank you so much.
Hi Idalba,
This is what the NEB site says.
"The enzyme is active in the pH range of 7.0 - 10.0, with optimal activity at pH 9.2 for both RNA and DNA substrates. Cleavage preferences have been observed at sites rich in adenylate, deoxyadenylate or thymidylate (1). Both DNA and RNA are degraded to 3´ phosphomononucleotides and dinucleotides. " Additionally, I have successfully used the protocol in Article Rapid and inexpensive preparation of genome-wide nucleosome ...
for DNA digestion.
Thanks Thilini for the information, I've been checking and the concentration of salts is critical for the enzyme, I must use a medium with less than 100 mM of salts, but the pH remains above 8, so it's best to use only PBS and/or or the enzyme buffer.
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