Etoh wash - confessions of a paranoid researcher....

[Michael G. Kurilla]

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.
> 
> Cheers
> 
> DAJ
> 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.

Michael Kurilla