I have a plasmid sample (10kb) with two different isoforms:
I want to make sure my sample has only nicked plasmids, no plasmids with a gap. Is there any way to verify this visually, or any enzymes that will remove gapped plasmid, but leave the nicked plasmid intact?
One possibility rather than removing gapped plasmids would be to repair or fill-in the gaps using e.g. Pfu DNA polymerase under conditions where strand displacement does not take place (as in Agilent's Quikchange site-directed mutagenesis kits, for instance).
Hope it helps!
For in gel visualization, you might treat your samples with S1 or Mung Bean nucleases (S1 is better). These nucleases will efficiently convert gapped plasmid into the linear one. Linear plasmid will migrate much faster in comparison with nicked form.
To remove gapped plasmid from your prep, you might teat your samples with 1) S1 nuclease, and then with 2) Lambda-nuclease. The first treatment will convert gapped plasmid into the linear one. The second will degrade linear plasmid.
Just an idea:
1) New England Biolabs sells SSB protein (Single-Stranded DNA Binding Protein) :
M2401S 500 µg/ml 50 µg $159.00 ;
2) Add it to the mix with plasmid
3) gapped plasmid would bind it (them) and would aquire high molecular wight
nicked plasmid would not
4) Run agarose or acrilamide gel at approriate (pH , salt etc. to ensure stability of protein/plasmid complexes)
I hope you will see the deference (two bands)
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