Talk:Knight:Beta-galactosidase assay/96 well format: Difference between revisions
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#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 μL 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 μg/mL -> 3.90625 μg/mL -> 1.953125 μg/mL -> 0.9765625 μg/mL | -> 7.8125 μg/mL -> 3.90625 μg/mL -> 1.953125 μg/mL -> 0.9765625 μg/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 μL of cell suspension to the first well | |||
#Add 175 μL of previous well to next well. | |||
#Add 175 μL of EZ Rich Media to that well. | |||
#Repeat dilution series until the well's solution looks totally clear. | |||
#Add an addditional well of 175 μL 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 12:57, 24 October 2007
A420 versus o-nitrophenol concentration
- 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
- Make up 1mL of 4 mg/mL ONP.
- Add 350 μL of 4mg/mL solution to the first well.
- Move 175 μL of previous well to next well.
- Add 175 μL of background solution to that well.
- Repeat dilution series until the well's solution looks totally clear.
- Add an addditional well of 175 μL background solution.
- Measure A420 and A550 of each well in the plate reader.
- 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
- 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 μL of cell suspension to the first well
- Add 175 μL of previous well to next well.
- Add 175 μL of EZ Rich Media to that well.
- Repeat dilution series until the well's solution looks totally clear.
- Add an addditional well of 175 μL EZ Rich Media.
- Measure A600 of each well in the plate reader.
- Plot A420 versus dilution factor. This relationship should be linear.