Mung Bean Nuclease for removal of ssDNA oligos
Posted 30 November 2011 - 09:39 AM
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!!
A rather confused and baffled postdoc in London
Posted 01 December 2011 - 09:52 AM
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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...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.