Ghost bands on plasmid preps

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Thu Apr 5 08:45:11 EST 2001


In <20010405093410.31073.qmail at ww02.jatek.com>, Daniel MacArthur wrote:
>
>John:
>
>> That sure sounds like the infamous "form 4" DNA. It is resistant to 
>> most restriction digestion. Make sure that you're not leaving the 
>> prep in the alkaline lysis step for too long, that's where this band 
>> comes from. It is irreversibly denatured, and won't cut, but can 
>> actually transform bacteria. In my experience, keeping the time 
>> from addition of the alkaline SDS to the addition of the neutralising 
>> solution less than five minutes (preferably less than four minutes) 
>> seems to minimize this ghost band.
>
>OK - I think next time I'll try cutting down the length of the lysis step or
>eliminating it altogether (another poster in this forum had some great
>suggestions regarding alternatives that I'm quite keen to try out).


Generally, once the alkali lysis solution has cleared you can go on to the
neutralization step.



>
>Now this might be a stupid question, but what exactly is meant be the term
>"irreversibly denatured"? When I read Paul Hengen's 1996 article on ghost
>bands [1] (derived from discussions on this newsgroup) I was confused by his
>description of the bands as "double-stranded, cyclic, coiled DNA composed of
>two intertwined, but permanently denatured, single strands of plasmid DNA".
>What is the chemical basis for this denaturation? Is the DNA covalently
>modified? If not, what stops it from reforming dsDNA? If it is covalently
>modified, how can it still transform bacteria?

I don't think anybody really knows for sure (OK, maybe I should say I don't
know). My guess is that it's not covalently modified, but kinetically trapped in
a conformation unfavorable to re-annealling. The bugs have a lot enzymes for
dealing with messed up DNA, so that can probably unwind it and fix it if you
give it to them.

I've seen it myself on occasion, and it's true that it doesn't seem to cut with
any restriction enyme. Interestingly, I never saw it in the old days of CsCl
preps; I guess it doesn't band it the bouyant density of ccc superciled DNA.

Nick

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu




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