EtOH + PBS = precipitation?
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.
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
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