Talk:Knight:Beta-galactosidase assay/96 well format

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Line 8: Line 8:
#Move 175 μL of previous well to next well.
#Move 175 μL of previous well to next well.
#Add 175 μL of background solution to that well.
#Add 175 μL of background solution to that well.
-
#Repeat until the well's solution looks totally clear.
+
#Repeat dilution series until the well's solution looks totally clear.
-
#Measure A<sub>420</sub> of each well in the plate reader.
+
#Add an addditional well of 175 &mu;L background solution.
 +
#Measure A<sub>420</sub> and A<sub>550</sub> of each well in the plate reader.
 +
#Plot A<sub>420</sub> versus ONP concentration.  This relationship should be linear.
Concentration series:
Concentration series:
Line 16: Line 18:
-> 7.8125 &mu;g/mL -> 3.90625 &mu;g/mL -> 1.953125 &mu;g/mL -> 0.9765625 &mu;g/mL
-> 7.8125 &mu;g/mL -> 3.90625 &mu;g/mL -> 1.953125 &mu;g/mL -> 0.9765625 &mu;g/mL
 +
 +
==A<sub>600</sub> versus cell density==
 +
#Grow an overnight culture to saturation in EZ Rich Media
 +
#Pellet the cells
 +
#Resuspend in 1/4 of the original volume with EZ Rich Media
 +
#Add 350 &mu;L of cell suspension to the first well
 +
#Add 175 &mu;L of previous well to next well.
 +
#Add 175 &mu;L of EZ Rich Media to that well.
 +
#Repeat dilution series until the well's solution looks totally clear.
 +
#Add an addditional well of 175 &mu;L EZ Rich Media.
 +
#Measure A<sub>600</sub> of each well in the plate reader.
 +
#Plot A<sub>420</sub> versus dilution factor.  This relationship should be linear.

Revision as of 15:57, 24 October 2007

A420 versus o-nitrophenol concentration

  1. Make up a solution of 7 mL to control for background absorbance in β-galactosidase assays
    • 800 μL permeabilization solution
    • 6 mL substrate solution without ONPG
    • 200 μL EZ rich media supplemented with kanamycin and AHL
  2. Make up 1mL of 4 mg/mL ONP.
  3. Add 350 μL of 4mg/mL solution to the first well.
  4. Move 175 μL of previous well to next well.
  5. Add 175 μL of background solution to that well.
  6. Repeat dilution series until the well's solution looks totally clear.
  7. Add an addditional well of 175 μL background solution.
  8. Measure A420 and A550 of each well in the plate reader.
  9. Plot A420 versus ONP concentration. This relationship should be linear.

Concentration series:

4mg/mL -> 2mg/mL -> 1mg/mL -> 0.5 mg/mL -> 0.25 mg/mL -> 0.125 mg/mL -> 62.5 μg/mL -> 31.25 μg/mL -> 15.625 μg/mL

-> 7.8125 μg/mL -> 3.90625 μg/mL -> 1.953125 μg/mL -> 0.9765625 μg/mL

A600 versus cell density

  1. Grow an overnight culture to saturation in EZ Rich Media
  2. Pellet the cells
  3. Resuspend in 1/4 of the original volume with EZ Rich Media
  4. Add 350 μL of cell suspension to the first well
  5. Add 175 μL of previous well to next well.
  6. Add 175 μL of EZ Rich Media to that well.
  7. Repeat dilution series until the well's solution looks totally clear.
  8. Add an addditional well of 175 μL EZ Rich Media.
  9. Measure A600 of each well in the plate reader.
  10. Plot A420 versus dilution factor. This relationship should be linear.
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