RNA Question

Cathy Sprankle Sprankle at ciit.org
Sun Jun 30 20:49:38 EST 1996


In article <elauzier-2606961558300001 at mac111.peps.ulaval.ca>,
elauzier at fse.ulaval.ca (Edouard Lauzier) wrote:

> When I use The Chomczynski and Sacchi method for RNA extraction...
> 
> 1. The homogenizer will shear DNA,  but why the latter goes to the
> interface and/or the organic phase ?  Since it does not shear the RNA, the
> "sheared"  DNA must be approximatly the same size.  What affects the DNA
> that doesn't affect the RNA  ( Na acetate ?, phenol pH ? ... ) ? 
> 
> 2.  When they say resuspend the pellet with 75% ethanol do they mean
> vortex,  just a little shake, good wash etc ?  Is it a critical step and
> why ?
> 
1.  Can't help you here--I just know it works, but don't know why.

2.  I usually vortex the sample pretty good at this point, and a colleague
of mine actually takes a P1000 with an RNAse free tip (critical!) and uses
the tip to mash up the pellet and pipet it up and down.  You're obviously
not going to get the RNA to go into "solution" (nor do you want to!), but
the more you can disperse it at this point, the easier time you'll have
getting it into solution in water.  Also I have found that if you don't
break up the pellet real well, it tends not to stick to the bottom of the
tube after the next spin--a real pain if you're trying to drain the
pellet.

Good luck!
Cathy

-- 
Catherine Sprankle
CIIT
e-mail:  sprankle at ciit.org
Opinions expressed (such as they are!) are strictly mine and do
not reflect those of CIIT.



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