b-gal assay

[Stephen Mamber]

In article <42k8gs$1m7 at eldborg.rhi.hi.is>, eiriksig at rhi.hi.is (Eirikur Sigurdsson) says:
>
>Hi.
>I am having a problem doing my b-galactosidase assay.  I am trying to measure
>lacZ transcription from a promoter I have fused in front of the gene.  On 
>X-gal I get fine blue colonies.  I am using the method of Miller, but using
>chloroform and SDS instead of toluene to rupture the cell membrane.  My
>problem is that the chloroform seems to "kill" all my b-galactosidase.  When
>I use only SDS I get yellow color after several hours (between 10 and 100 
>units), but when I also use the chloroform I do not get any colour at 
>all.  I thought this might be because of the instability of the fusion protein
>but I get almost the same results measuring b-gal activity from a wild-
>type strain.  There I get a little yellow colour when I use chloroform, but
>much less than if I only use SDS.  Also the b-gal activity I get from the wild-
>type strain, using just SDS, is much to low.
>
>Does anybody have any idea what might be wrong?  
>Any help would be very much appreciated.
>
>
>
>Eirikur Sigurdsson                      "Nothing improves an innovation
>Institute of Biology                     like lack of controls."
>University of Iceland
>
>eiriksig at rhi.hi.is
>
Why are you fooling around with solvents?

My protocols are based on  the following refs:  Elespuru, R. K., 
and R. J. White.  1983.  Biochemical prophage induction assay:  
A rapid test for antitumor agents that interact with DNA.  
Cancer Res. 43:2819-2830, and  Elespuru, R. K., and M. B. Yarmolinsky.  
1979.  A colorimetric assay of lysogenic induction designed for screening
potential carcinogenic and carcinostatic agents.  Environ. Mutagen. 
1:65-78.

I start with 40 ul cells+media in the wells of microdilution trays.  
Cold ZCM buffer (120 ul/well) is followed by o-nitrophenyl-b-
D-galactopyranoside (Sigma), 4 mg/ml in medium A (25 ul/well).  
(ZCM buffer is composed of Na2HPO4.7H2O, 16.1 g; KCl, 0.75 g; 
MgSO4.7H2O, 0.25 g; b-mercaptoethanol, 2.7 ml; chloramphenicol, 
25 mg; and deionized water, 1 l.  The buffer is adjusted to pH 7.0, 
filter-sterilized and stored at 4 C.  Medium A contains K2HPO4, 
10.5 g; KH2PO4, 4.5 g; (NH4)SO4, 1 g; sodium citrate dihydrate, 
0.5 g; and deionized water, 1 l.  Medium A was sterilized by 
autoclaving at 121 C for 15 min).  After incubation at 37 C for 
30 min., 50 ul of cold, filter-sterilized Na2CO3, 1 M, are added 
per well to stop color development.  Absorbance is measured at 414 nm.

SWM

mamber at synapse.bms.com