Talk:Knight:Beta-galactosidase assay/96 well format: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
Line 22: | Line 22: | ||
#Continue the assay as described at [[Knight:Beta-galactosidase assay]]. | #Continue the assay as described at [[Knight:Beta-galactosidase assay]]. | ||
#Plot the β-galactosidase activity in Miller Units as a function of the A<sub>600</sub> of the culture. | #Plot the β-galactosidase activity in Miller Units as a function of the A<sub>600</sub> of the culture. | ||
Is there a better way to do this using a plate reader? | |||
==A<sub>420</sub> versus [[ONP|o-nitrophenol]] concentration== | ==A<sub>420</sub> versus [[ONP|o-nitrophenol]] concentration== |
Revision as of 13:26, 24 October 2007
This is an outline of various control experiments that I need to do. It is a work in progress and has not been done.
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.
β-galactosidase activity versus A600 of culture
- Grow an overnight culture of MG1655 and R2000.E0433
- In the morning, dilute back both samples into 1mL of EZ Rich Media to an A600 of 0.001 (via a Nanodrop reading).
- Do samples both with and without 1mM IPTG.
- Prepare many 1.5mL eppendorf tubes with 80 μL of permeabilization buffer.
- Every hour, measure the A600.
- At each hour point, take three 20 μL samples of culture and add it to 3 tubes.
- Continue the assay as described at Knight:Beta-galactosidase assay.
- Plot the β-galactosidase activity in Miller Units as a function of the A600 of the culture.
Is there a better way to do this using a plate reader?
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