Hi ..I run an agarose gel on a plasmidic extract and I have different bands. I don't know if they are different forms of the same plasmid or different plasmids. I want to determine if the bands are different plasmids and, if yes, what band is the plasmid that carry the genes of resistance. In your opinion, how can I do these two things? Does it make sense doing a Southern Blot on the gel with plasmidic extract, by using the resistance gene as a probe? Can you describe a little bit the steps I should follow for this? I have never done it before.. I know that I could transform cells with my plasmidic extract to determine the resistance plasmid but I am struggling doing it, so I am looking for an alternative method to get this info.In your opinion, can I then cut the band corresponding to the resistance plasmid, purify it and use it for transformation experiments?
Any suggestion would be really appreciated.
Thanks
I can't give you any advise on the Southern blot part but maybe on the separation by transformation. I assume you have a plasmid free bug that is able to propagate your plasmids and is able to express the resistance gene. Transformation of multiple plasmids happens at a lower frequency than transformation of a just one plasmid. Let the cell grow on an antibiotic free agar after transformation and then transfer single colonies on an antibiotic containing agar by picking with a pipette tip. Do plasmid preps from colonies from antibiotic free and antibiotic agar and analyze by gel.
Do you use a kit for plasmid isolation? The reason you see more than one band on the gel could be too harsh lysis condition during the prep. Look here: http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/. So basically it could be all the same plasmid - just different conformations.
hello,
1) you could design and synthesize primers for common resistance genes in your strain or species, there are plenty literature described nowadays thanks to the next generation sequencing projects.
2) how many bands do you see? remember that 3 bands could be the same plasmid in three diferent conformations, supercoiled, circular and linear.
3)how to know if this is the case and you have only one plasmid? you purify this bands and perform restriction enzyme protocols, if you get the same bands of digestion means that it was the same plasmid, diferent digestion patterns will get you the exact number of different plasmids you might have.
4) besides that, run PCR experiments in all of this bands and check if they have some sort of resistance genes.
5) it would be by then time to correlate plasmid species and resistance.
6) go buy yourself a treat because it is a lot of work :)
pd. If you have plenty money send this plasmids to sequencing, it would give you the exact number of plasmids and the resistance genes.
- Badges
- Science topic