Megaplasmid best extraction protocol - (Nov/07/2013 )
I am a bioinformatician so not much wet lab experience but have identified a putative megaplasmid using mapping of reads from sequencing and was wondering what is the best method of megaplasmid extraction for confirmation? Don't have direct access to PFGE which would be best and normal plasmid extraction kits aren't showing anything as expected. Plasmid should be ~100kb. Any help would be appreciated.
You could try a conventional genomic DNA extraction and then see if you can resolve it on standard gel electrophoresis, but it will be tricky. PFGE would certainly work better. You could also try putting it through something like FPLC.
A field inversion gel would resolve a 100Kbp easily. You don't need a full blown pulse field gel system.
We found that more than half of the denatured plasmid DNA could not find and re-anneal with its partner strand after being denatured during alkaline lysis. So we developed a potent non-alkaline lysis method for plasmid extraction and turned out it improved pDNA yield many folds. Your megaplasmid, same for BAC, might be even harder to re-nature after alkaline lysis and they may get lost like genomic DNA in the cell debris after neutralization. I think our non-alkaline lysis based AquaPlasmid could help. If you are interested at trying it out, feel free to contact us.