Talk:Knight:Beta-galactosidase assay: Difference between revisions

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(New page: ===500mM dibasic sodium phosphate=== 7.098g Na<sub>2</sub>HPO<sub>4</sub> in 100mL H<sub>2</sub>O ===1% hexadecyltrimethylammonium bromide (CTAB)=== 100mg CTAB in 10mL H<sub>2</sub>O ===...)
 
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100mg CTAB in 10mL H<sub>2</sub>O
100mg CTAB in 10mL H<sub>2</sub>O


===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.
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===1M sodium carbonate===
===1M sodium carbonate===
*52.995 g Na<sub>2</sub>CO<sub>3</sub> in 500mL H<sub>2</sub>O
*52.995 g Na<sub>2</sub>CO<sub>3</sub> in 500mL H<sub>2</sub>O
==Measuring repression as a function of A<sub>600</sub>==
#Grow an overnight culture of P20060.E0433
#Dilute back to A<sub>600</sub> = 0.001 (as measured on the Nanodrop) in two tubes (1mL each)
#Let grow for 1 hour
#Prepare the permeabilization solution and aliquot out 80 &mu;L per microfuge tube.
#Add 20 &mu;L of 50 mM IPTG to both tubes and 1 &mu;L AHL to one tube.
#Every hour, measure the A<sub>600</sub> and add 20 &mu;L to 3 microfuge tubes.
#Proceed with the [[Knight:Beta-galactosidase assay]] as normal.
#Plot the Miller Units as a function of A<sub>600</sub>.

Latest revision as of 17:36, 6 February 2008

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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