Mammalian Lysis buffer for B-gal Assay

J.A.H. Hendriks jaco.hendriks at gironet.nl
Sun Aug 29 03:14:06 EST 1999


Mimi Liao <m.liao at student.unsw.edu.au> schreef in berichtnieuws
3.0.1.32.19990820122254.00795a20 at pop3.student.unsw.edu.au...
> Hi all,
>
> I am currently lysing the CHO-K1 cells with lysis buffer containg 50 mM
> Tris-Cl (pH8.0), 150mM NaCl, and 1 % Trioton-100. And i'm measuring the
> B-gal content using ONPG colormetric assay. when i added the Na2HCO3 to
> stop the reaction, the solution became very cloudy. However, the solution
> cleared up o/n.
>
> My questions are;
> 1) Do we really need to add the stop solution ? Can we just measure the
> absorbance straight away without stopping the reaction ?
> 2)Are there any recipe for mammalian lysis buffer which are compatible
with
> B-gal assay ?
>
>
>
> Thanks very much in advance

I allways do a chloroform extraction and a 10 minute spin after stopping the
color-reaction. Everything has to be done in the same way and I think this
would be very hard if you don't stop the reaction.
Good luck
Brenda





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