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#Allow plate to return to room temperature before re-reading at 560:660 nm. These are the final readings.
#Calculations
##For both initial and final readings, subtract the absorbence absorbance values obtained at the 670 nm wavelength from those obtained at the 560 nm wavelength.
##Subtract the initial absorbance reading from the final reading, then calculate sample concentrations from the standard curve.
#To each well, add 3-8 uL of serum samples and appropriate volume of standards (3 if you expect a lot of lipolysis, and greater volume if you expect less).
#Incubate plate at 37 deg C for 5 minutes (or room temperature for 30 min).
#launch Gen5 Software on the computer #Choose Glycerol Assay protocol #Under the procedures menu, choose wavelength 540 nm and set the shaking orbital for 2 seconds prior to reading the plate#Under the layout menu specify what wells you would like to be read #Read the plate at 540 nm. This is the initial reading.(if the readings do not seem correct, check for air bubbles and reread the plate)#Save the file
#To each well, add 20 uL of triglyceride reagent. Incubate plate at 37 deg C for ~5 minutes. The triglyceride reagent contains a lipase that breaks down the triglycerides into glycerol and fatty acids. The assay measures the glycerol in the sample, so the first reading tells us how much glycerol is present.
#Re-read the plate at 540 nm. This is the final reading. The second reading tells us how much glycerol is present after the triglycerides present in the serum have been broken down.
#Save the file
#export both files onto excel
#Calculations
##Subtract the initial reading from the final reading. This tells us what proportion of the glycerol in the sample came from triglycerides.
##For each of the initial, final and calculated absorbances, calculate the concentration of the samples from the standard curve.