i cut a plasmid by HPAI enzyme (creation blunt end) and dephosphorylate simultaneously. after run on gel, 2 band observed.
i nead to linearized plasmid for cloning.
How can I decipher between linear and circular plasmid on gel?
Linear plasmid will be the expected molecular weight. Circularized will not- it will either be supercoiled, and run fast, or nicked. If you Google this you should be able to find a pic with the usual three bands, and a detailed explanation.
my plasmid is synthetic . this plasmid is not replicated in bacteria.
i should be able to find a pic with the usual three bands?
http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
There's a decent explanation. I'm not sure if a synthetic plasmid will behave the same way, though. However, linearized plasmid will run at the appropriate molecular weight. So, if you see two bands, hopefully one is at the correct length. If you aren't sure, do a digestion timecourse for a diagnostic gel. You should see one band get larger, and the other band get smaller as everything get linearized.
Good luck!
Make sure your plasmid has one HpaI restriction site. If it has two sites for HpaI restriction, a complete digestion will give two bands when ran on an agarose gel.
The circular linear plasmid runs faster than the circular plasmid.If you have a circular plasmid it means that the restriction enzymes were unable to digest the plasmid.
Hi there,
I think you have only partial digest so you have both linear and circular forms. Extend the time of digest and unique band should be observed.
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