My plasmid size is ~6000bp, when I extracted the plasmid and run it in 1% agarose, it shows one band above 1kb, one band in between 1kb and 7000bp, and one band in between 5200bp - 4000bp. There is none band appear on the exact bp of my plasmid (i.e. 6000bp). May I know is this possible? Why does this occurred? Thank you.
This article has some good explanations. https://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
If you linearize the plasmid before running the gel, you will get one nice band at the right size.
Did you linearise your plasmid before running ?
If uncut multiple bands often not quite the right size simply represent different DNA conformers with different amounts of supercoilung
To accurately size DNA it must be linear
Dear Utsa
I think you make an interesting point but DNA uncut can manifest as 3 different conformers which will always make size interpretation more complicated than a single linear band of exactly the correct size
What is more the problem with multiple bands is that you can never be completely sure if the picture is further complicated by contamibation/or degradation so I would contend that simple digestion to linearise is always perspicuous and best
If your plasmid is not subject to nicks and there fore closed covalent circularised as you say supercoiled ladder will work well
However cutting to eliminate supercoiling and associated multiple bands will work fine with well prepared DNA
https://www.researchgate.net/deref/https%3A%2F%2Fbitesizebio.com%2F13524%2Fhow-to-identify-supercoils-nicks-and-circles-in-plasmid-preps%2F
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