world

Stephen R. Lasky Stephen_Lasky at brown.edu
Fri Apr 8 09:13:39 EST 1994


In article <2o2isfINNfk5 at jhunix.hcf.jhu.edu>, pchen at welchlink.welch.jhu.edu
(PING CHEN ) wrote:

> 
> Dear bionetters,
> 	I have been trying to prepare nuclear extracts from promonocytic cell
> lines (U1, U937) for a while and have not been very successful. I used 
> Dignam's protocol or the modified version of the protocols that other people 
> used successfully for making nuclear extracts. My problem is that I could not
> get a majority of the cells to lyse without breaking the nuclei as judged by 
> 

We have made extracts from monocytic cells (U937, HL-60 and several CML
cell lines) using this tech.  No problems.

> (1) whole cells were still present after homogenization with a glass Dounce 
>     homogenizer - after over 20 strokes;

What Pestle are you using?  Are you swelling the cells in hypotonic buffer?
 Do you use Trypan Blue exclusion to determine lysis?  If you are using the
tight pestle and swelling the cells, you should be able to get 90% lysis
with  10 strokes as determined by trypan blue staining.

> 
> (2) the "nuclear pellets" get very viscous when I tried to resuspend them, 
>     which I take as an indication that DNA was out in solution. 

The pellets are alway viscous in our hands and we have had no problem with
the extracts. Everybody who I have spoken to gets viscous nuclear pellets.

> 
> The protocols seem straight forward and many people reference them in the
> litereature.  I do not know if I am missing some important hints.  I would
> appreciate it if anybody who reads this message and has experience and is 
> willing to help a desperate grad.student.
> 

The main question is do your extracts work?  You don't say what you are
using them for.  

> Ping Chen
> 
> pchen at welchlink.welch.jhu.edu

-- 
***************************************************************
Stephen R. Lasky, Ph.D.       Brown University/Roger Williams Medical
Center
e-mail: Stephen_Lasky at brown.edu         LandLine: 401-456-6572
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A nuclear war could ruin your whole day.
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