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After double digest my plasmid is 2Kbp shorter! - (Sep/22/2006 )

this is really weird (as my whole research is last months rolleyes.gif ):

my vector is 6.6Kpb i digest with each one alone EcoRV and BglII and i get correct size band.
i double digest and the size of the band is around 3.5 Kbp... blink.gif i purify and it gets bigger to around 4Kpb. blink.gif

there is no site between the enzymes so size shouldn;t differ. buffers are all the same used. please solve this puzzle for me. smile.gif


where do these enzymes cut?
its probable that the two sites are very close so that u will not be able to distinguish between the two bands.


Am I correct to say that the entire 3.5kb band suddently looked to become 4kb? This is not a situation of an appearance of a 4kb band alongside the normal 3.5kb band.

If my understanding is correct, I would like to know if this observation is repeatable? Is so try repercipitating the DNA, and resuspend it in water (take good care of the DNA if it is in water). Make sure all the salts are gone. And run the gel again. This sounds like DNA retardation due to salt. Maybe it is?


This is a strange one...

My first idea is that the 3.5 kb band you see when you double digest is actually two 3.5 kb bands co-migrating (2 x 3.5 kb = 7 kb, which is approximately 6.6 kb). This would mean that the two sites for your enzymes are on opposite sides of the plasmid -- cut singly with either enzyme, and you get a linearized plasmid; cut with both enzymes simultaneously, and you get two fragments of approximately equal size which co-migrate in the gel as a single band.

A second possibility is that both enzymes are failing to cut in the double digest, and the 3.5 kb band you see is actually uncut plasmid, which, due to it's compact size relative to linear DNA, migrates more quickly, and appears to be 3.5 kb when compared against linear standards.

You can diagnose the first possibility by cutting the plasmid with one enzyme, gel purifying the fragment, and then cutting it with the second enzyme. If this produces a 3.5 kb band, then your sites are seperated from one another by half the size of the plasmid, and your 3.5 kb band comprises two fragments that are co-migrating.

You can detect the second possibility by cutting the plasmid with both enzymes, then recovering your 3.5 kb fragment from the gel, and cutting it with only one of the enzymes. If this produces a 6.6 kb band, then your 3.5 kb band was uncut plasmid, and the problem is that the double digest is not working.