EtOH + PBS = precipitation?

LU Pin bolupin at uxmail.ust.hk
Wed Feb 28 12:17:14 EST 1996


Colin Rasmussen (colin at fungus.biochem.ualberta.ca) wrote:
: LU Pin wrote:
: > 
(quoated message deleted)

: Sounds  like the PO4 is precipitating. What is the final % EtOH you 
: are trying to achieve, and why have you decided on PBS as opposed to 
: other buffers.

: Colin

Thank you for your responses. I forgot to say in my last post that 95%
ethanol + 5% 160mM NaCl seems ok. 

Why prepare 95% ethanol + 5% PBS: 
I am analysizing the synchronization of mammalian cells by measuring the DNA
contents using flow cytometer. I got a protocol from one of my colleages.
Briefly, cells are washed in PBS, then fixed in 95% ethanol. Cells
will be stained by propidium iodide subsequently. And I was told that the
95% ethanol should be prepared from abs. ethanol in PBS. That means 95%
ethanol + 5% PBS. Then I have this problem. Now I prepare 95% ethanol in
160 mM NaCl. From the reply I got, it seems no way to avoid precipitation
in 95% ethanol/PBS.

I've searched around the literatures and found that the common way to fix
cells is 'drop-wise' addition 5 ml of 95% ethanol into 2 ml cell suspension
in PBS. How to prepare the 95% ethanol was not metioned. If somebody has
the experiences, would you please share with me? Thank you.


     _\\\ ///       Lu, Pin		Department of Biology
     \ __   /       Hong Kong University of Science & Technology
      C  @~@        Phone: 2358-7312    e-mail: bolupin at uxmail.ust.hk
          >         Fax:   2358-1559    WWW:    http://www.ust.hk/~bolupin/





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