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

John Nash nash at biologysx.lan.nrc.ca
Wed Sep 1 09:43:14 EST 1993


In article <1993Sep1.074325.19613 at gserv1.dl.ac.uk> daj at uk.ac.ic.nhm ((David Johnston) daj) writes:
>From: daj at uk.ac.ic.nhm ((David Johnston) daj)
>Subject: Re: Etoh wash - confessions of a paranoid researcher....
>Date: Wed, 1 Sep 1993 08:40:57 GMT


>>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).

I know mixing will help, but I haven't mixed in years, just added the 70% 
ethanol, and then sat it on the bench for a moment, then centrifuged it for 
60 sec, decanted supernatant, and used.  Two different things I do.. I 
aspirate off the supernatant with a drawn out Pasteur pipette end (hooked 
up to a water-line vacuum).  You never lose pellets that way - at worst, 
they stick to the glass tip.  It gets out much more of the ethanol.  Also, I 
do a final rinse in absolute ethanol. It seems to dehydrate the pellet, so 2-
5 min on the bench is enough to dry out the pellet before adding the TE or 
water or whatever.

  cheers, John

  John Nash                           | Email: Nash at biologysx.lan.nrc.ca.
  Institute for Biological Sciences   |
  National Research Council of Canada | Email to my other NRC accounts
  Ottawa, Ontario, Canada.            | is usually forwarded here.
	  *** Disclaimer:  All opinions are mine, not NRC's! ***



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