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How does circular, uncut pGEM-T migrate? - (Aug/31/2011 )


Anybody knows how does circular, uncut pGEM-T (with fragment of course) migrate on agarose? How many bands does it yield at at what level of the Ladder?



Assuming there is no linear or supercoiled DNA then it should result in 1 band only. If you just have a standard plasmid extraction you may see 3 bands depending on the integrity of the DNA you have extracted. These bands will be the supercoiled, the nicked (i.e. circular, single strand breakage) and the linear (double strand break). If you see more bands then there has been further digestion of the DNA, or you have degraded genomic DNA or RNA in your prep.


...or if isolated from a recA positive strain you'll see plasmid multimers (two/three/four/etc-times the vector size).