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nicked linear DNA

how does it run on a gel

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5 replies to this topic

#1 theo22

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Posted 30 June 2013 - 05:07 AM

When I run a plasmid on a agarose gel, it will appear as three bands: linear, supercoliled (which runs faster then linear) and nicked (which runs slower then linear. But what happens to nicked linear DNA (a fragment of e.g. 1 kb). How will the nicked form run compared to the linear unnicked form? Will it run more slowly? And is it possible to discriminate those two forms on a agarose gel? Or on an acrylamide gel?

Thanks for your reply

#2 bob1

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Posted 30 June 2013 - 12:28 PM

The point about nicked and unnicked DNA is that the plasmid is circular - nicked DNA unwinds and runs as the purely circular form, not linear. Linear fragments will separate based on their size. I don't think that you will be able to distinguish a single base break in one strand for linear fragments unless you run on a acrylamide gel and have very small fragments.

#3 theo22

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Posted 01 July 2013 - 01:44 PM

My 1 kb fragment contains 6 nicks in a region of about 60 bp, all in the same strand. After denaturation, this 60 bps will be removed from this strand. Thus the resulting product will be double-stranded DNA of 1kb with a stretch of 60 bp where the DNA is single stranded. Any chance that this will be visible on a gel as a shift compared to the wt fragment?

#4 bob1

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Posted 01 July 2013 - 05:01 PM

Probably, though I doubt that you will detect it easily on an agarose gel. You might be better off trying PA gels or using a system like the "gel on a chip" sort of things (like this one).

#5 phage434

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Posted 01 July 2013 - 06:36 PM

Could you cut the dsDNA fragment so that the relevant region is shorter? This would make the difference more obvious.

#6 theo22

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Posted 18 July 2013 - 12:27 PM

I tested some things and I found that I can see a very clearly shift of the 1 kb PCR fragment on a 6% polyacrylamide/TBE gel after adding 5 single strand nicks. And surprisingly, the shift can also be detected with a 6kb nicked PCR fragment.




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