I ran uncut plasmid with my insert and digested, what will be the reason for the difference as the uncut plasmid consist of the insert and the digested plasmid released the insert, so the band length of uncut will be greater than linear? Why in get pic it shows less? Please see the attached gel image...and give your valuable suggestion. Ladder start with 250 bp and vector is ptz57 R/T
an intact vector with,or without insert is circular and under tension so its lowest energy conformation is twisted and knotted . There can even be 2 or more stable conformations making it look like 2 or 3 bands of dna on a gel. Once the insert is cut out the tension is destroyed and only one conformation ( more linear) is produced and because this is a different shape from the circular it runs at an apparently different size but this can be either larger or smaller than the uncut plasmid. This is entirely normal
an intact vector with,or without insert is circular and under tension so its lowest energy conformation is twisted and knotted . There can even be 2 or more stable conformations making it look like 2 or 3 bands of dna on a gel. Once the insert is cut out the tension is destroyed and only one conformation ( more linear) is produced and because this is a different shape from the circular it runs at an apparently different size but this can be either larger or smaller than the uncut plasmid. This is entirely normal
As Paul said it s quite normal.. it is all depending on the conformation of the DNA molecule and its friction with agarose gel while migration. normally you see 3 or 2 bands in uncut plasmid lane because of different conformations of isolated plasmid regarding the quality of isolation... supercoiled, linear and open circular are the conformations respectively you see from the bottom to top in the gel image... because of their conformation they have different migration speed, so supercoiled is the fastest because of less friction in agarose gel. supercoled and OC can not be used to judge the length of the plasmid so you can not compare your digested plasmid with them...
As Paul and Saber has mentioned, you have got correct result. never compare the size of a linear molecule with that of circular one(supercoiled form). your uncut plasmid has good amount of super coiled form that has high mobility due to its compact structure and nicked form with low mobility. both these forms are of same size,( mobility difference is due to conformation). when you digest both forms get digested and become linear- this you can compare with the marker for the size.
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