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"Nicked" open circular plasmids --- Incomplete digestion?

"Nicked" open circular plasmids --- Incomplete digestion? - Molecular Cloning Forum

"Nicked" open circular plasmids --- Incomplete digestion? - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 09-15-2011, 02:13 PM
Pipette Filler
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Question "Nicked" open circular plasmids --- Incomplete digestion?



I have been trying to digest my pUC plasmid with a single enzyme --- HindIII.

However, the digestion did not seem to proceed to completion even after 3 hours.

I observed 3 bands (from fastest to slowest):
Supercoiled, linear (cut) and another which appears to run really slowly. I ran the uncut plasmid and did not observed the "slow" band. Thus, I believe it came up due to the RE reaction.

I looked at various forums and people have suggested that the "slow" ones are actually "nicked" open circular plasmid arising from incomplete digestion. However, I have doubts about this --- Can type II endonucleases really "nick" strands? I thought the homodimers would cleave 2 strands at a time, and incomplete digest will result not having all the recognition sites "cutted", rather then leaving "nicks"?

If they are not "nicked" open circular plasmid, what could they be? Will that be a cause of concern, especially when I am having problems digesting all the plasmids.

Any suggestions?
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circular , digestion , hindiii , incomplete , incomplete digestion , nicked , nicked dna , open , plasmids


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