Dear colleagues,
has somebody any tip to minimize plasmid multimerisation during growth of bacterial culture (E. coli, high-copy plasmid Bluescript). I would like to isolate whole plasmid only in its monomeric superhelical form for subsequent use in EMSA.
I know Topoisomerase IV, which is able to decatenate plasmid multimers, but this is quite expensive way for me.
Also I know it is possible to excise monomeric bands from gel, but this is very time-consumable way and yield is quite low.
I am interested in some elegant trick to say bacterial culture to produce only monomers :-)
Maybe some growth additive exists? What about different concentration of NaCl in growth medium? Any other tips?
Thanks for all answers
Martin
Probably most of the bands you're seeing aren't from plasmid multimers but are from different supercoiling states of the same monomeric plasmid.
https://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
I think there are some multimers in recA- strains but they're a pretty low percentage of the plasmid present as far as I am aware.
Before going through with any of the more complicated stuff to try to totally remove the other supercoiling forms post-extraction, I'd try reducing the amount of lysis time on your plasmid preps, maybe centrifuge the neutralized lysate at 4° if you haven't been doing that already. The reason these forms happen as far as I know is that the lysis procedure during plasmid prep is pretty harsh and will damage some of the plasmid DNA no matter what, but the amount varies depending on a lot of factors including how long you keep the DNA in the strongly alkaline conditions of the lysis buffer before neutralizing.
But the only non-enzymatic way I personally am aware of that will get you close to 100% super coiled plasmid DNA is a cesium chloride gradient, which I've never done but they don't sound fun to prepare and require an ultra-centrifuge.
Probably most of the bands you're seeing aren't from plasmid multimers but are from different supercoiling states of the same monomeric plasmid.
https://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
I think there are some multimers in recA- strains but they're a pretty low percentage of the plasmid present as far as I am aware.
Before going through with any of the more complicated stuff to try to totally remove the other supercoiling forms post-extraction, I'd try reducing the amount of lysis time on your plasmid preps, maybe centrifuge the neutralized lysate at 4° if you haven't been doing that already. The reason these forms happen as far as I know is that the lysis procedure during plasmid prep is pretty harsh and will damage some of the plasmid DNA no matter what, but the amount varies depending on a lot of factors including how long you keep the DNA in the strongly alkaline conditions of the lysis buffer before neutralizing.
But the only non-enzymatic way I personally am aware of that will get you close to 100% super coiled plasmid DNA is a cesium chloride gradient, which I've never done but they don't sound fun to prepare and require an ultra-centrifuge.
If you don't need it to be circular, you can just cut it by a single cutting restriction enzyme. All the supercoiled forms (plus the band of the nicked form - the second above the major supercoiled band) will just transform into the same linear band.
Otherwise, the presence of superheical multimers is stated to be strain dependent, you can try several different E. coli strains. Generally, you cannot get rid of the nicked circuar form, it will always accompany the bright superhelical band (monomeric).
Though, I've seen a paper on selective precipitation of superhelical DNA. Maybe it will be useful if you want to remove the nicked form: Article Condensation of supercoiled DNA by MnCl2
if you are sure that these are multimers, not supercoiled, changing bacterial strain or using low OD bacterial growth for plasmid Isolation would be optional.
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