Talk:Knight:Beta-galactosidase assay

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(Measuring repression as a function of A<sub>600</sub>)
Current revision (20:36, 6 February 2008) (view source)
 
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100mg CTAB in 10mL H<sub>2</sub>O
100mg CTAB in 10mL H<sub>2</sub>O
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===10% sodium deoxycholate===
+
===1% sodium deoxycholate===
-
100mg sodium deoxycholate in 1mL H<sub>2</sub>O
+
10mg sodium deoxycholate in 1mL H<sub>2</sub>O
Light-sensitive.  Store at 4&deg;C.
Light-sensitive.  Store at 4&deg;C.

Current revision

Contents

500mM dibasic sodium phosphate

7.098g Na2HPO4 in 100mL H2O

1% hexadecyltrimethylammonium bromide (CTAB)

100mg CTAB in 10mL H2O

1% sodium deoxycholate

10mg sodium deoxycholate in 1mL H2O

Light-sensitive. Store at 4°C.

1M sodium carbonate

  • 52.995 g Na2CO3 in 500mL H2O

Measuring repression as a function of A600

  1. Grow an overnight culture of P20060.E0433
  2. Dilute back to A600 = 0.001 (as measured on the Nanodrop) in two tubes (1mL each)
  3. Let grow for 1 hour
  4. Prepare the permeabilization solution and aliquot out 80 μL per microfuge tube.
  5. Add 20 μL of 50 mM IPTG to both tubes and 1 μL AHL to one tube.
  6. Every hour, measure the A600 and add 20 μL to 3 microfuge tubes.
  7. Proceed with the Knight:Beta-galactosidase assay as normal.
  8. Plot the Miller Units as a function of A600.
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