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Plasmid digestion create a bigger plasmid

Ecl136II plasmid digestion troubleshooting

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#1 ringlin

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Posted 01 November 2011 - 07:31 AM

Hi

I've got a problem with my plasmid digestion for cloning.
I want to create a linear plasmid with blunt ends to ligate my inserts from the phusion PCR.

But their is the problem, if I digest my plasmid (5.8kb) with Ecl136II i get lines at 4kb, 5.8kb and >10kb.
How can a plasmid double the size by digestion. Mean I get an output but a to low. With 5ug on a 50ul reaction input i get 25ng/ul output.
The picture in the appendix shows the plasmid and a 1kb marker. For purification I cut out the main line but you see the hole.

Here are some other inforamtions:

Purification gel: 1% agarose
Enzyme is new
Buffer is also new
Enzyme is fast digestion from Fermantas
Tried also shorter or longer incubation
tried also with or without inactivation

Has anybody had the same problems or do you have an idea what i'm doing wrong?

Thanks for your ideas

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  • pCSC5-Ecl.jpg


#2 pDNA

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Posted 01 November 2011 - 01:44 PM

there are three possible conformations that plasmid DNA can have on an agarose gel:
1) supercoiled form ...most compacted runs at a smaller size than the plasmid actually has (in your case 4 kn) ...no strand of the DNA is nicked
2) linear form ...double strand is nicked ...plasmid runs at the size you actually expect (in your case 5.8 kb)
3) open circle form ...only one strand is nicked ...plasmid runs much higher than expected (in your case >10 kb)

in your case it is hard to say if it is really open circle form that you see at >10 kb or residual genomic DNA or plasmid multimers ...normally i'm used to very minor bands that appear for the open circle and in your case it is a really strong band ...so i'm not quite sure what we have hear. To me it seems more likely that these are plasmid multimers (plasmid isolated from a recA+ strain?) or residual gDNA.

Overall it seems that the restriction digest was only partial since you have a lot of DNA left in the supercoiled form ...so you should extend digestion time or units of enzyme or both.

Regards,
p





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