HELP ! get DNA (< 500bp) out of gel
bernard at elsie.nci.nih.gov
Sat Jun 15 15:13:19 EST 1996
In article <4ptrli$nib at mark.ucdavis.edu>, ez026048 at rocky.ucdavis.edu says...
>I had problem of isolating small DNA fragments from gel. I had tried
>promega "Wizard" DNA purification system and Bio 101 Geneclean product.
>Both did not end up satisfied result. I have no problem, however, in
>isolating longer DNA fragment (eg >1000 bp) using the same methods above.
I used to use the Promega "Magic" PCR preps/Clean-up resin with good
results but the "Wizard" formulation is definitely worse for me (10 - 50%
of the yield as gauged by a side-to-side comparison). I then switched
to Qiagen's QIAquick which gave yields that were lower than the Magic
but consistently higher than the Wizard. I still use the QIAquick
method when I need reasonably good recovery from non-low melt gels.
My current standard is the home-made Celite formulation posted
to this newsgroup ("Merlin" prep) which is essentially the same as the
old "Magic" kit. This has worked well for me for fragments 0.2 - 7 kb
and yields cuttable, sequencable, transcribable, PCR'able DNA in good
yield (eg. estimated 50 - 80% from low melt TAE gels).
I believe that Paul has summarised his favourite extraction
methods in the FAQ/website/TIBS article etc. (I don't have access to
any of these at this very moment so I can't give you a direct pointer).
While I am on the subject, can any other users of the Merlin
protocol confirm my suspicion that, when used for plasmid maxipreps,
the diatomaceous earth does not bind nucleases (I appear to get good
preps from HB101 and GM2163) as opposed to the current Wizard/silica
gel resin which most certainly does. Promega now instruct you to bind
the crude TE extract to the resin for the briefest possible time to
minimise the carry over whereas I've shaken crude TE from HB101 with
the Merlin resin for 30 minutes and then eluted nuclease free plasmid.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
"If I knew what was going on I'd really be indignant." -Crow T. Robot, MST3k
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