Miller Units definition

M.J. Horsfall mjhf at troi.cc.rochester.edu
Fri Mar 5 21:58:45 EST 1993


In <2B97CE04 at adminbldg.lan1.umanitoba.ca> GIETZ at bldghsc.lan1.umanitoba.ca writes:

>Howdy!
>        Could someone please tell how to 
>calculate Miller Units for the ONPG beta gal assay.
>I have been looking for a copy of Experiments in Genetics
>by J. Miller (which is always referenced) but no one around 
>here seems to have it!!! No papers I have
>looked at give a good description of how to do this calculation.
>All  papers  lead back to this book.  Which I cannot
>get easily (very frustrating).  Thanks in advance!
>________________________________________
>Dan Gietz
>Department of Human Genetics
>University of Manitoba

Briefly, the method is as follows:

1. Start with your culture (grown in minimal medica supplimented with 20
ug/ml B1, 0.001M MgS(O)4 and 0.4% glucose, plus 0.001M IPTG) on ice 
( for ~ 20 min). You should already know the OD(600) of this culture 
-should be around 0.28-0.70 (=2-5 X 10^8 cells/ml).

2. Add aliquots of the cultures to the assay medium (Z buffer, given below).
Final volume = 1 ml. For high levels of B-gal try 0.1 ml cells, for low
levels try 0.5 ml.

3. Permealize the cells by adding one drop of toluene -vortex for 10 sec.
Evaporate the toluene off -try agitation for 1/2 hr at 37 degrees.

4. Incubate tubes at 28 degrees for 5 min. Add 0.2 ml 4 mg/ml ONPG (in 0.1M
phosphate buffer, pH 7.0) -mix. 

5. Stop the reaction with 0.5 ml 1M (Na)2(CO)3 when you think its done (nice
shade of yellow) or over a time course.

6. Take OD(420) and OD(550). OD(420) should be in the range 0.6-0.9. OD(550)
is used to correct for cell debris. The latter can be omitted if you spin
down your reactions just prior to measuring absorbance.

The formula is:
               OD(420) - [1.75 * OD(550)]
Units = 1000 X --------------------------
                    t * v * OD(600)

       -where: [1.75 * OD(550)] is the correction for light scattering by
               cell debris at OD(420). o-nitrophenol conc. is measured at
               OD(420).

                t = time of the reaction (min)

                v = volume of original culture used (ml)

A fully induced E. coli culture grown on glucose will show ~ 1000 units, and
an uninduced culture about 1 unit.


Z buffer: (for 1 litre)

0.06M (Na)2HP(O)4-7(H)2O
0.04M Na(H)2P(O)4-(H)2O
0.01M KCl
0.001M MgS(O)4-7(H)2O
0.05M B-mercaptoethanol
-adjust to pH 7.0
-do not autoclave


I hope this helps,

Mike
--
M.J. Horsfall --------------------------------- mjhf at troi.cc.rochester.edu
Department of Biophysics                        --------------------------
University of Rochester ----------------------- Phone:(716)-275-8703 (Lab)



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