Miller Units definition

Donald Chen - Microbiology chend at
Fri Mar 5 23:43:42 EST 1993

In article <2B97CE04 at> GIETZ at 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
	strain dependent.
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,

	Don Chen
	Oregon State University


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