I amplified some plasmids using chemically competent DH 5 alpha as per the standard protocol. I routinely verify the band pattern of midiprep product by agarose electrophoresis. This time i got some unusual bands. For some of the plasmids the bands have shifted upward to higher kb position. I have been extremely careful to avoid cross contamination so i didn't handle any other plasmid during this experiment. What could be the reason for this strange band pattern? valuable suggestions will be highly appreciated.
P.S: The plasmids harbor lysine to arginine point mutation so all the plasmids move at same position
upper image - Plasmid samples i used for transformation
lower image - Midiprep product
Dear Poshan,
The larger plasmid bands (lanes 3, 5 & 8) in your lower gel may simply be plasmid dimers - this can often occur in the cell, though most of the time they are quickly resolved back into the normal monomeric form of the plasmid (you should be using a recA strain to minimise this sort of problem). You should be able to easily check this using a restriction endonuclease - dimers and monomers will produce the same fragment pattern after digestion.
It may be that some other form of recombination (perhaps involving a small deletion) may have produced these larger plasmid bands, and in general, you should avoid using them if at all possible.
Regards, Andrew.
Dear Poshan,
The larger plasmid bands (lanes 3, 5 & 8) in your lower gel may simply be plasmid dimers - this can often occur in the cell, though most of the time they are quickly resolved back into the normal monomeric form of the plasmid (you should be using a recA strain to minimise this sort of problem). You should be able to easily check this using a restriction endonuclease - dimers and monomers will produce the same fragment pattern after digestion.
It may be that some other form of recombination (perhaps involving a small deletion) may have produced these larger plasmid bands, and in general, you should avoid using them if at all possible.
Regards, Andrew.
When plasmid DNA is isolated and run on an agarose gel, you may observe 2, 3 or even 4 or more bands. You can see in the diagram, how these forms will show up on an agarose gel in terms of relative migration speeds. DNA found in the supercoiled form is not easily accessed by replication machinery. During replication, cellular topoisomerases nick one strand of the DNA helix and relax the superhelical tension, thus allowing polymerases to gain access to the DNA. Using the rubber band analogy, nicked circle DNA is the rubber band without any twists introduced. This large floppy circle is the slowest migrating form in an agarose gel.
Regrettably, I do not agree with Dr. Spiers's opinion, because in the case of dimer or trimer and so on, we don't expect to see such sharp bands but we expect to see smear. Because the multimerization for all of the molecules does not occur uniformly.
and also I don't agree with Dr. Goraichuk because there is a general shift in some lanes, and the different forms of any plasmid isn't the subject at all!
Instead, I believe that there was a probable error in the process of making constructs so that incorrect fragments have been inserted in vectors.
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