miniprep problem - (Mar/19/2008 )
Hello everyone
I isolated my plasmid with QIGEN miniprep after that I check the size I loaded around (100ng) from the plasmid the problem is I have an upper band in the top is there any way to avoid it
And this is the image
This is completely normal - in fact I would be concerned if you didn't see more that one band (this would indicate it had been cut or nicked).
The different bands you are seeing are simply differently coiled versions of the plasmid.
Because each time I did the isolation the upper band decrease
So is there any way to decrease the upper band I try several things but I could not find?
Hi,
As ailedroc said, it's normal to see few bands, the upper one correspond to circular, middle is the nicked, and bottom is the supercoiled.
However there are ways to avoid "damaging" the isolated plasmid:
(1) Lysis time should not exceed 5'
(2) Don't vortex after lysis solution had been added. Just gently invert tubes multiple times to homogenize. This is to avoid fragmenting genomic DNA and also large plasmid DNA.
(3) Use fresh culture. Don't leave cell pellet in -20C and work on plasmid isolation later. Though I've done that without problem, but sometimes, I do see more nicked than supercoiled.
If supercoiled DNA is needed, I've came across purification of supercoiled from non-supercoiled using using differential centrifugation. Another approach would be enzymatic using DNA gyrase.
Bests
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So is there any way to decrease the upper band I try several things but I could not find?
Not yet, but the answer is quite patentable.
I think open and nicked conformations of plasmid are present in bacteria at any time. This is especially true if the plasmid is to be transcribed, copied and all. If it existed as supercoiled, I don't think the conformation is accessible for the above-mentioned processes.
As mentioned, you can subsequently do a CsCl centrifugation to isolate your supercoiled plasmid from other forms. Or you can try enzymatic treatment such as DNA gyrase, in which the method has been patented somewhere.
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With DNA gyrase will take more time to do it for each isolation but could you please explain more or protocol about CsCl
Centrifugation
And thank you very much
Why do you need to decrease the upper band? If you look at intensities, most of your DNA is supercoiled.
Because give variation when I transfect so I try to decrease
celljunctions.med.nyu.edu/lab/notprivate/methods-html/CsCl.html
www.oardc.ohio-state.edu/stockingerlab/Protocols/Plasmid%20DNA%20Prep.pdf
If you're really interested to minimize variation of transfection, you could try linearizing the plasmids before transfection. Anyway, depending on the exp, there's a way to "normalize" the variation. E.g. gating in flow cyto.