plasmid isolation help urgent.. - (Mar/20/2007 )
hi, i m trying to isolate plasmid from azospirillum spp. i dont know how big the plasmid is. literature says, the plasmid may be even upto 9 mb.. megaplasmids.. but, when i do the isolation according to the usual mini prep protocol for bacterial samples, i get absolutely no bands in the agarose gel. (.7%)
i suspect this may be bcos the size is too big and hence pulse field electrophoresis has to be performed. or bcos i use only distilled water to prepare buffers, the distilled water might contain DNAse.
Do any of u think there may be some other problem? pls do reply asap.
If what you said is true, this is almost a chromosome. You cant isolate it without breaking it into 20-40 kb fragments. You may need to lyse it in agarose plug and run it with pulse field electrophoresis.
genehunter-1 is correct.
Agarose plugs, pulse field gel and followed by gel electrolution is the only way. 9mb is as larger then many yeast chromosomes.
It's larger than all yeast chromosomes, and it will be challenging to separate even with pulsed field gels.
I don't think it qualifies as a plasmid.
It is so large. Do you using a kit to isolate? If you are, no bands is right.
Maybe you can isolate it like chromosome.