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This protocol is directly derived from Sean Moore's Beta-Galactosidase Assay (A better Miller). Please go there for the original protocol. This protocol just contains specifics regarding stock solutions I've made.



See Talk:Knight:Beta-galacosidase assay for stock solution recipes.

Permeabilization Solution

For 2mL:

(You need 80 μL per sample.)

Substrate solution

For 10mL

(You need 600 μL per sample.)


  1. Grow cultures under whatever conditions you wish to test.
  2. During growth
    1. Make permeabilization solution.
    2. Pre-measure 80 μL aliquots of permeabilization solution into 1.5 mL microfuge tubes and close them.
  3. Measure Abs600 of cultures and RECORD IT!
  4. Remove a 20 μL aliquot of the culture and add it to the 80 μL of permeabilization solution.
    • The sample is now stable for several hours. This allows you to perform time-course experiments.
    • Also include a blank (solutions-only) sample for zero'ing the spec later.
  5. Make substrate solution.
  6. Warm samples and substrate solution to 30°C
  7. Start timer counting up.
  8. Every 15 secs, add 600 μL of substrate solution to a sample tube.
  9. Note the time of addition.
  10. After sufficient color has developed, add 700 μL of 1M Na2CO3, mix well.
  11. Note the stop time.
  12. Once all reactions are complete, transfer the tubes to a microfuge and spin for 10 minutes at full speed.
  13. Gently remove tubes from centrifuge.
  14. Measure the absorbance at 420nm and 550nm. (Use UV-Vis protocol on Nanodrop).

Calculate Miller Units as:

1000 * \frac{(Abs_{420})}{((Abs_{600} \text{ of culture sampled})*(\text{volume } [0.02 \text{ mL}])*(\text{reaction time}))}


1000 * \frac{(Abs_{420} - 1.75*Abs_{550})}{((Abs_{600} \text{ of culture sampled})*(\text{volume } [0.02 \text{ mL}])*(\text{reaction time}))}


Comments on the assay


Error fetching PMID 7538113:
  1. Jeffrey H. Miller. A short course in bacterial genetics. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, 1992. isbn:0879693495. [Miller-1992]
  2. Error fetching PMID 7538113: [Zhang-JBC-1995]
    (from which this assay was derived)

  3. [by] Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory, 1972. isbn:0879691069. [Miller-1972]
    (original Miller assay)