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Plasmid Purification and Digestion Problem

plasmid digestion restriction enzyme supercoil

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#1 Emrah

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Posted 02 October 2012 - 12:06 PM

Hi there;

I purified an unknown plasmid from an unknown bacteria. It is a low copy plasmid and very hard to purify. After purification I only observe a single band. From literature I know that plasmids can form 3 different shapes (supercoil, released circle, full length linear). Do you think is there a problem about purification or is it totally normal?

Because of sequencing, I have to digest it with restriction enzyme. But when it comes to digestion, all of the enzymes that I used fragmented the plasmid into pieces (I use EcoRI, HpaII, Pst1). Is there a known restriction enzyme for cutting espacially low copy plasmids?

Thank you very much....

#2 bob1

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Posted 02 October 2012 - 12:33 PM

The copy number does not matter once it is isolated - it's just DNA then, so there are no specific restriction enzymes. The patterns of fragements you got should tell you certain things about the plasmid (I'm not telling you what, as this looks suspiciously like a homework problem).

#3 Nayeem991

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Posted 02 October 2012 - 07:01 PM

You can isolate the plasmid in large volume (midi method, following the protocol mentioned in molecular cloning by maniatis et al). you will get higher amount of plasmid. Where do you get the single band in the gel (size of your undigested band)? do you get isolated band after digestion or it just become smear? If it is smear i am afraid the band you see is the band of chromosomal DNA because generally we see 3 bands for undigested plasmid.





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