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Mung Bean Nuclease for removal of ssDNA oligos


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#1 Dr_Patel

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Posted 30 November 2011 - 09:39 AM

Hello all,

Was wondering if anybody has ever used Mung Bean Nuclease to remove ssDNA oligos from an annealing reaction. I have annealed my fwd and rev oligos to generate dsDNA oligo and am using this in a protein/peptide pulldown assay. I wanted to make sure any protein pulldown with the dsDNA was due to the dsDNA that I have annealed and not to residual ssDNA left unannealed. I am thinking of treating my annealed material with mung bean nuclease. Do you think this is possible? aIf so, does anybody have a protocol for chopping up ssDNA oligo using this nuclease? I have tried digesting just some bogstandard ssDNA oligo (20bp) but dont see much cleavage on an agarose (3%) gel. I still get one single band running at the same size as the uncut oligo. Should I expect some sort of laddering or smearing or total removal of band if I digest with this enzyme?? If anybody out there could help me it would be much appreciated!!

Thanks!

From,
A rather confused and baffled postdoc in London

#2 hobglobin

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Posted 01 December 2011 - 09:52 AM

At least Mung Bean Nuclease only cleaves single stranded DNA. So it should work. Length of fragments should be no problem.
No idea why your test did not work, but 20bp on an agarose gel is anyway difficult, because it's then usually a broader smear-like band.
The supplier should have a protocol in their application notes or on their website (if not google for a protocol). But usually with the correct buffer, temperature and sufficient time it should work, if the enzyme is okay.

Forgot to mention: S1 Nuclease is an alternative.

Edited by hobglobin, 01 December 2011 - 09:55 AM.

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