fragment isolation w/o glass milk?

nishir at ohsu.edu nishir at ohsu.edu
Tue Aug 9 11:46:53 EST 1994


In article <325dbaINN2ceh at rs1.rrz.Uni-Koeln.DE>
DSCHLIEP at CIPVAX.BIOLAN.UNI-KOELN.DE (Daniel Schlieper) writes:
>
>Dear netters,
>
>recently Wang & Rossman published how they isolate DNA fragments from 
>agarose gel by centrifugation (Nucleic Acids Research, 1994, Vol. 22,
>No. 14, pp 2862-2863). They use a 0.5 ml tube with a hole at the bottom, 
>fitted inside a 2 ml tube and filled with glasswool, Sephadex G-10 and
>the smashed DNA-containing agarose gel (up to 0.6% agarose).
>
>I have two questions:
>- Are there similar, probably even easier methods? For gels with higher
>  concentraitons of agarose than 0.6% ?
>- What is the best method to wash out the ethidium bromide?
>
>Looking forward to answers, Daniel.
>
>Daniel Schlieper (DSCHLIEP at GENETIK.UNI-KOELN.DE)
>Institut f r Genetik, Universit t K ln, Weyertal 121, D-50676 K ln, Germany 
>
We use a modification of a method that was posted here about a year ago by "The
End" (name escapes me, sorry) in Indiana which is easier (only 'cause I don't
like to mess around with glass wool and cutting tubes).  You run your digested
DNA on a low melting point agarose gel (whatever percentage you need to get
good separation).  Cut out your band, put it into a microfuge tube, melt it at
65-70 degrees, rapid freeze (put it in liquid nitrogen or alcohol/dry ice, or
just stick it into the -80 freezer), thaw at 37 degrees, and spin in microfuge
(12-15,000 g) for 10 min.  Collect supernatant and use for ligations or for
template in nick translation.  For some reason this stuff doesn't get labeled
very well when using random priming.  If you want to precipitate the DNA to
concentrate it, then add 1/10 vol 3M Na Ac before adding 2 vol 100% ETOH.  Try
to minimize exposure to UV while cutting to get best ligation efficiency (we
always cut on a semi-UV permeable glass plate (the old EM plates for photos
work very well)).

Rae Nishi
OHSU
Portland, Oregon




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