Lidstrom: Molecular Devices Plate Reader
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- Machine = molecular devices: spectramax 190
- Software = Softmax Pro, version 5.3. (manual)
Order of reagents
- add 20-40uL cell extract per well. Add ~180 uL of mix of substrate & cofactors dissolved in buffer. Adding the large volume to the small volume should yield decent mixing.
Should I run a whole plate at once or rows/columns at a time?
- There are a few reasons you want to read the wells you load as quickly as possible. As soon as you mix all your reagents and cell extract, your reaction starts. If your assay has a non-linear reaction rate at short times (often the case) then you will lose some information at early timepoints. For example, often a reaction is fastest at small times, and slows down as the reaction proceeds and products build up.
- Unless you know your assay is highly linear for 30+ seconds, Janet recommends you run one cell extract with every substrate/no-substrate mix per scan of the machine, then move to the next one. OR, run all of your cell extracts with one substrate/no-substrate mix at a time.
Do I need to use the crystal plate or a plastic plate?
- NADH assays are run at 340 nm. This is right on the lower boundary for what wavelengths are suitable for our plastic so it shouldn't matter much. If the crystal plate is around, you might as well use it. At a minimum you will generate less trash.
- If kinetic data needs to be exported and you can't get the data exported as one column per well:
- Go to Settings --> Preferences
- Change the settings to Time
- Your files should look like this:
- Note: This assay ran for < 1 minute and Microsoft excel misinterpreted the times as minutes, not seconds. This may not be a problem if you run your assay longer, but it was a problem here.
- (from manual)
- The machine can report Vmax in units of milli-Units/min or Units/second. What are "Units" you ask? It is just the slope. Multiply by 1,000 to get d(Absorbance)/min.