Etoh wash - confessions of a paranoid researcher....
Michael G. Kurilla
mgk2r at Virginia.EDU
Wed Sep 1 11:59:00 EST 1993
daj at uk.ac.ic.nhm writes:
> >When you do an ethanol wash of a DNA precipitate do you try to resuspend the
> >pellet and spin, rinse the pellet and spin, rinse the pellet and just dry...
> You'll never truely resuspend the pellet 'cos its a precipitate, but you
> may dislodge it from the tube wall and break it into bits if it is big and
> you vortex it like hell. Also, often, even with gentle handling, 70%
> ethanol appears to dislodge pellets from the tube wall. So.....
> (1) If you can see your pellet clearly and it is still attached to the wall
> after addition of the 70% and a gentle mix/vortexing (remember, the aim of
> the rinse is to help flush out salts so mixing must help) then there is
> probably no need to spin, but if your pellet is small/invisible/loose then
> what is the "cost" of an extra minute or two in the microfuge against peace
> of mind (as the DNA is already precipitated into "lumps" (whether you can
> see them or not) you only need to do a brief respin).
> (2) For the record, I always vortex and respin after addition of 70%, I
> always position the tubes with their cap hinges in the same orientation in
> the micofuge so I know where to expect the pellet to be and I always
> transfer the alcohol washes into a petridish so that I can retrieve the
> pellet if it does still come away. OK so I am paranoid but I rarely loose a
> pellet. For the hyper-paranoid out there I believe that NYCOMED do a
> microfuge tube which has been specifically treated to hold onto
> precipitated DNA more firmly than standard polypropylene tubes. I haven't
> tried them ........yet.
> David A. Johnston
> Dept of Zoology, The Natural History Museum, Cromwell Road,
> South Kensington, London SW7 5DB.
> (tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)
After seeing all the discussionon EtOH rinses, I wonder exactly
why people rinse with 70% EtOH. Are you removing salt that has
precipitated or are you diluting the salt present in the
residual liquid before drying?
I believe it is the latter (unless the initial salt
concentration was very high. For minipreps, we have stopped
rinsing after precipitation. Rather we aspirate the
water/isopropanol then quickspin in a centrifuge. The volume
of residual liquid is about 40 - 50 micrliters that is then
aspirated, dried in a Savant ,and resuspendeed to 50 microliters
with TE. We have never had problems cutting even with multiple
low salt REs.
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