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BISC110/F12: Series 3 Experiment 9 Hill Reaction: Difference between revisions
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BISC110/F12: Series 3 Experiment 9 Hill Reaction (view source)
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'''Varying the Reaction Rate:''' The Hill reaction occurs at different rates depending on the light intensity and the quality and concentration of the thylakoids. Our goal today is to determine the best light intensity and thylakoid concentration for a 90 sec. reaction. You will measure the reaction rates by taking absorbance readings over that time period in 15 s intervals. We will use the drop in absorbance over time as a measure of reduction of blue DCPIP to colorless DCPIPH. If we can control all the variables properly, this reduction rate should also be an indication of the rate of photosynthesis since the light and dark reactions are coupled. <br><br> | '''Varying the Reaction Rate:''' The Hill reaction occurs at different rates depending on the light intensity and the quality and concentration of the thylakoids. Our goal today is to determine the best light intensity and thylakoid concentration for a 90 sec. reaction. You will measure the reaction rates by taking absorbance readings over that time period in 15 s intervals. We will use the drop in absorbance over time as a measure of reduction of blue DCPIP to colorless DCPIPH. If we can control all the variables properly, this reduction rate should also be an indication of the rate of photosynthesis since the light and dark reactions are coupled. <br><br> | ||
Depending on the number, distribution (clumped or not) and condition of thylakoids in your reaction tube, you may obtain varying reaction rates with a given light intensity. Therefore, you will test your thylakoids neat (undiluted) and diluted 1/2 with resuspension solution. We measure the incident light using a light meter in units of µmol photons m-2 s-1. <br><br> | Depending on the number, distribution (clumped or not) and condition of thylakoids in your reaction tube, you may obtain varying reaction rates with a given light intensity. Therefore, you will test your thylakoids neat (undiluted) and diluted 1/2 with resuspension solution. We measure the incident light using a light meter in units of µmol photons m-2 s-1. <br><br> | ||
Typically, the light intensity is set at ~80 µmol photons m-2 s-1 in order to drive the reaction at the desired rate—a drop in absorbance per 15 s that is consistent throughout a 90 s trial. However, that intensity may not be appropriate for all thylakoid preparations at all concentrations so you will have to experiment with both the light intensity and the concentration of your thylakoid suspension to find a rate that does not use up all of our substrate (DCPIP) too quickly (before 90 sec.) or that is too slow to approach substrate depletion in two minutes. The 15 s reading intervals consists of 10 s of thylakoid illumination and 5 s to read the absorbance in the spectrophotometer. Your goal is to establish conditions favorable for a 90 s experiment. This data collection at interval of 15 s should yield 7 data points (i.e., absorbance readings at time = 0, 15, 30, 45, 60, 75, and 90 s) that are all in the linear portion of a curve when you graph absorbance (y axis) vs. time (x axis). If your light intensity is too high and/or your thylakoids too concentrated, your curve will flatten before the 90 second reading because you have depleted your DCPIP substrate and those data points after substrate | Typically, the light intensity is set at ~80 µmol photons m-2 s-1 in order to drive the reaction at the desired rate—a drop in absorbance per 15 s that is consistent throughout a 90 s trial. However, that intensity may not be appropriate for all thylakoid preparations at all concentrations so you will have to experiment with both the light intensity and the concentration of your thylakoid suspension to find a rate that does not use up all of our substrate (DCPIP) too quickly (before 90 sec.) or that is too slow to approach substrate depletion in two minutes. The 15 s reading intervals consists of 10 s of thylakoid illumination and 5 s to read the absorbance in the spectrophotometer. Your goal is to establish conditions favorable for a 90 s experiment. This data collection at interval of 15 s should yield 7 data points (i.e., absorbance readings at time = 0, 15, 30, 45, 60, 75, and 90 s) that are all in the linear portion of a curve when you graph absorbance (y axis) vs. time (x axis). If your light intensity is too high and/or your thylakoids too concentrated, your curve will flatten before the 90 second reading because you have depleted your DCPIP substrate and those data points after substrate is limiting will be unusable. If your light intensity is too low and/or your thylakoids too dilute or of poor quality, the data points obtained within an optimal 90 sec. reaction will not measure enough of the linear part of the reaction to give a reliable rate. <br><br> | ||
'''Measurement of Hill Reaction Rates:''' For each reaction tube, do the following in turn.<br> | '''Measurement of Hill Reaction Rates:''' For each reaction tube, do the following in turn.<br> | ||
#Label a 13mm glass test tube 1/2. Using your P200, add 100 microliters (0.1ml) resuspension solution. Mix the stock '''thylakoid suspension''' gently by flicking bottom of tube. Using a 1ml pipet (NOT your P200!) ,gently draw up and add 100 microliters of thylakoids to the equal volume of resuspension solution. Mix gently and keep both the stock and 1/2 dilution tube of thylakoids on ice. | #Label a 13mm glass test tube 1/2. Using your P200, add 100 microliters (0.1ml) resuspension solution. Mix the stock '''thylakoid suspension''' gently by flicking bottom of tube. Using a 1ml pipet (NOT your P200!) ,gently draw up and add 100 microliters of thylakoids to the equal volume of resuspension solution. Mix gently and keep both the stock and 1/2 dilution tube of thylakoids on ice. | ||
