Help

Rick Bright rbright at mnw.net
Thu Dec 19 20:51:25 EST 1996


Rick Bright wrote:
> 
> I have been using a protocol successfully to extract DNA from blood.  At
> one point, I use a low salt buffer to salt in the proteins, coupled with
> SDS.  I then add a high salt (5M NaCl) to precipitate the proteins and
> follow with a spin to pellet them, leaving the DNA in the salt
> solution.  As of yesterday, after this spin, it is not pelleting.  I
> have the clear salt solution layer, but it is on the bottom of the tube,
> while the "reddish" goo layer of the proteins is floating on top of the
> salt solution.  How can this protein layer now have a lower density than
> the salt/DNA solution?
> 
> 1) Has my low salt buffer gone bad?  It was made on 11/18, consists of
> TRIS/EDTA/0.4MNaCl, MgCl2, KCl.  It seems it would be stable for a long
> while, I keep it refrigerated.
> 
> 2) Has the SDS gone bad?  10%, I keep refrigerated and warm to rt before
> use.  Does the cool, thaw cycle hurt it after a while, does it go bad
> after a while?
> 
> I would appreciate any ideas.
> 
> Rick
> rbright at mnw.net

I have an idea, hopefully someone can confirm or reject.  I keep my
working buffers refrigerated and set them out about 30 min before the
extraction.  When I initially made them (pH specific at 7.6) they were
at room temp.  However, if I don't let them warm to room temp before
using, the pH is much lower (around 6.1).  This could have a serious
impact on my extraction procedure, I think.  

1) Does this sound like it could be the problem?

2) Do I need to keep the working buffers refrigerated?

3) Do the buffers go bad after a period, say 30 days or so?

I appreciate any feedback.

Thank you.

Rick Bright
rbright at mnw.net



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