denatured supercoiled dna - (Dec/06/2005 )
i think i've got a a denatured supercoiled dna band after my maxi-prep and it's resistant to restiction digestion.
Will it affect my subsequent expriment as i will generate nested deleted plasmid. i will need to do transformation after that, will this denatured dna goes into Ecoli?
If the alkaline lysis step is allowed to proceed for longer times or at higher temperatures, irreversibly denatured DNA will result. (Vinograd and Lebowitz 1966.) This material travels just below cccDNA on an agarose gel, stains poorly with ethidium bromide, and can comprise approximately 3% of the total DNA. This material is not cut by restriction enzymes, but it can transform bacteria. Thus it is a source of background in transformation that cannot be eliminated by extended restriction digestion or dephosphorylation. Treatment with T5 exonuclease, which possesses single-stranded endonuclease activity and 5’-exonuclease activity reduces the transformation efficiency of denatured DNA by two orders of magnitude. (J. R. Sayers as cited in Methods and Reagents 1996 TIBS 21 441-442.) This denatured DNA is thought to be double-stranded, cyclic, coiled DNA composed of two intertwined, but permanently denatured, single-strands of DNA.
Damn tfitzwater... that was a good answer.
Thanks for your fantastic answer.
So what is the exact pH, incubation time, and temperature before we see irreversible denatured DNA?
For more information on denatured DNA, see J. Vinograd and J. Lebowitz 1966. Physical and topological properties of circular DNA. J. Gen. Physiol. 49:103.