Hi all,
I am extracting plasmids from my bacteria. They are natural plasmids and I don't know the size, nor the sequence. I am trying to make plasmid profiles. I run the gel on the extracts in order to visualize the plasmids. I am able to see more bands in the gel. I was wondering how I can know if the bands I see correspond to the same plasmid with different conformations, or if they are different plasmids? And how can I know the number copy of the plasmid in the bacterium?
Thanks,
Silvia
Hello Sylvia,
If you can recover enough DNA from a gel slice, you could try to compare the restriction digests of the various species present in your samples. Labeling the digestion products with kinase and gamma-32P ATP may help to increase the sensitivity of detection.
Best wishes,
Pierre
Hello Sylvia,
If you can recover enough DNA from a gel slice, you could try to compare the restriction digests of the various species present in your samples. Labeling the digestion products with kinase and gamma-32P ATP may help to increase the sensitivity of detection.
Best wishes,
Pierre
Dear Sylvia,
If you want to isolate plasmid DNA, you crack your cells open and carry out a miniprep, trying very hard not to get any contaminating genomic DNA in your sample. If you want genomic DNA, you crack your cells open in a different way and try to isolate as much of the stuff as possible.
And to complement Pierre's comment, if you want to know their copy number the simplest path is to sequence them and then use qPCR (relative quantification) against a known chromosomal single-copy gene.
Hi Silvia,
Remember, isolated plasmids have 3 basic conformations: Supercoiled, Nicked, and Linear. Most likely what you are seeing is the same plasmid in it's different conformations. To test this, you can try and find an enzyme that cuts your plasmid only once (will have to be trial and error since they are naturally occuring plasmids). When cut, as you know, they will all linearize and run at the same speed through your gel. As far as copy number, this site gives a great run down on the calculations. If you quantify your bacteria prior to isolating plasmid, and assuming you have 100% lysis and 100% extraction, then you can determine number of molecules of plasmid per cell.
http://bitesizebio.com/20669/how-to-calculate-the-number-of-molecules-in-any-piece-of-dna/
Firstly,more highlight more possibility you want,secondly,compere the plasmid,it has open condition,thirdly,if you extract the plasmid too violence that you may broken the palsmid and some small fragments may have.The end,you should marking out and re-choose your bacteria
Restriction digestion of isolated plasmids and also try wit different antibiotics for their subculture, transfer each plasmid to competant cells and check for their quantification.
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