I compared my uncut plasmid to the same plasmid cut with a single restriction enzyme that is a single site cutter and the uncut plasmid ran in the gel slower. The size of my cut plasmid is around 3.7kb, which is the expected plasmid size, but the uncut plasmid is around 7kb. The uncut plasmid shows 3 bands, the brightest band around 7kb and the other two being above 10kb. Any explainations?
Hi.
This is indeed the normal behaviour of uncutted plasmid. It is all about supercoiling of DNA. Uncutted plasmid could be as open circle, supercoiled, etc..
On agarose gel electrophoresis, the covalently closed circular (ccc) form migrates faster than the linear form, the linear form (3.7 kb) faster than the open circular (oc) form (7kb). The above bands at 10 kb may be multimers? genomic DNA? contaminant plasmid? Presumably you do not succeed to obtain the ccc form. With mechanical shearing (with seringe and a curved needle) the supposed oc form at 7 kb would give the linear form (3.7 kb)
Article Simple procedure for distinguishing CCC, OC, and L forms of ...
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