Hi all,
I have to separate bacterial plasmids (after extraction) on agarose gel. Which electrophoresis conditions would you suggest me to use (in terms of hours, voltage)?
Thanks
There really isn't enough information to answer your question.
What size (in kb) are the plasmids?
What is your plasmid preparation method?
Are you running them intact or after digestion with restriction endonucleases?
Are the plasmids preps free from chromosomal DNA?
Hi there,
In complement of Deepan's post: provided the plasmid sizes are different enough, I think separating linearized plasmids is the most sensible approach to have as it will generate a single band per plasmid and the estimate of the size will allow to identify the plasmid on agarose gel. After purification a self ligation step would allow to recover the circular plasmids.
I don't know the size of plasmids. I am checking that the bacterium have them actually. In am running them intact after manual extraction with the Bennett method. I will not use a kit for that.
In that case you have to try several conditions. Remember that most "wild type" plasmids are very low copy number and quite large so you'll need to use large culture volumes. Ideally, if you have the capability for pulsed-field gels electrophoresis, use that.
Otherwise, you'll need quite long gels with low %agarose in TAE or TBE buffer run slowly at 4degC (5V/cm).
As an example see the attached, but if you do a search on Google Scholar you'll find 100s of early papers from the 50s-70s describing various methods for discovery of plasmids.
There's no magic formula for this and you'll have to carry out a systematic trial-and-error by getting early clues to the size and numbers of plasmids followed by optimised conditions to separate the on gels.
Good luck!
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