Miller Units definition
Donald Chen - Microbiology
chend at ucs.orst.edu
Fri Mar 5 23:43:42 EST 1993
In article <2B97CE04 at adminbldg.lan1.umanitoba.ca> GIETZ at bldghsc.lan1.umanitoba.ca writes:
> 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!
We've been using the following protocol:
1. grow cells to OD600 0.3-0.5 (or mid-log)
2. record OD600
3. to 1 ml cells, add 1-2 drops toluene (although some add CCHCl3)
shake/vortex, shake 30 min, 32 C
4. add 200 ul, 4mg/ml ONPG
5. wait until the color turns bright yellow (not dark yellow), record time
6. stop with 500 ul 1 M Na2CO3
7. record OD550 and OD420
8. Units= [OD420 - (1.75 x OD550)] / [(time in min) x OD600 x (vol in ml)]
then multiply by 1000
a. for me, the best cell OD600 seems to be around 0.5-0.8, but this is
b. I've used 1 ml of cell because then the vol variable is not a factor.
c. I use toluene because that's what I was taught in the beginning, but
another student used CCHCl3 with good results.
d. it seems to me, that the shaking is to both mix cells with the
membrane disrupter, as well as remove excess disrupter through
evaporation (my tubes aren't covered). also, I use plastic spec
tubes, and CCHCl3 scores tubes unless it's removed to some degree.
e. the ONPG is made fresh and diluted in 0.1 M phospate buffer.
f. if the color is too strong (eg dark), the OD values are so high that
I don't trust the readings (eg over 1.5-2.0). this is compounded by
the fact that the color intensifies over time even after stop is added.
g. you should do a few test runs with your controls as well as your test
strains, both to feel comfortable as well as understanding the difference
between your positive and negative controls.
Hope this helps,
Oregon State University
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