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Running undigested plasmids on gel


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#1 fiskarn

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Posted 10 September 2013 - 02:00 AM

Hi,

 

I am currently working with a plasmid with an insert separately mutated in 3 diffferent locations (giving 1 unmutated and 3 different mutated variants). When I run the undigested plasmids (4.5 kB with insert) on an 0.8% agarose gel the unmutated variant and 2 mutated variants separate identically with 1 supercoiled and 1 relaxed band each. However, the third mutated variant produces similar bands but way further up in the gel. According to the ladder these bands are twice as big as for the three other variants (I know size is difficult to judge with intact plasmids, but still..). When I digest the plasmids with single-cutters XhoI and HindIII from Fermentas I see single bands with the same size for all plasmid variants (and an extra small band for the insert when cutting with both RE simultaneously). So my question is - can the migration pattern for undigisted plasmids vary this much (dispite exactly the same bp lenght of the plasmids and only a few bp variance in sequence)? Or can the different migration be due to dimerization of my plasmid?

 

Super if someone could give me some input on this!

 

 



#2 phage434

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Posted 10 September 2013 - 04:58 AM

A rare, but possible event is the creation of double plasmids, twice the length of a normal plasmid. This can involve mutation of the plasmid ori. Doubled colE1 oris, for example, will not typically replicate well, but a single mutated ori version will. A "single" cutter will in fact cut these twice, and you will see the correct length linearized fragment. You can fix this, if you want, by single cutting, religating, and retransforming. This happens typically if the concentration of vector and insert DNA is extremely high during ligation, so aim for lower DNA concentration.



#3 fiskarn

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Posted 11 September 2013 - 09:56 PM

Thank you phage434 for your solid input. I think I will do as you suggest.






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