BISC110: Series 3 Experiment 9 Hill Reaction: Difference between revisions

1.  Materials: Each pair will have a ring stand with a test tube holder, and a fiber-optic illuminator with a 150W quartz-halogen projection lamp.  You will have to find the best arrangement to measure the reduction of DCPIP accurately and reproducibly.  A handheld light meter and timer will be provided.  Set up 5 ‘reaction tubes’ to begin with, adding 5mL of reaction mixture, but do not add the thylakoid membranes, which must be kept on ice.  Add these thylakoid membranes just before each reaction mixture is tested.  [Reaction mix: 50mM sodium phosphate, pH 6.8 buffer, 100mM sorbitol, 5mM MgCl2, 0.05mM DCPIP]

2. Blanking the Spectrophotometer ([[Appendix B]]):  Use a tube containing 5mL of resuspension solution and 50µL of your thylakoid suspension to blank the Spec20 at 580nm (filter level to the left).  With Parafilm® over the top of the tube, invert once to mix, then wipe the tube with a Kimwipe® before blanking the instrument.  Do not vortex.  You may need to prepare a fresh blank periodically.  

4. Measurement of Hill Reaction Rates:  For each reaction tube, do the following in turn and work as rapidly as possible and in dim light.  Mix thylakoid suspension gently by flicking bottom of tube.  Immediately remove 50µL of thylakoid suspension and add to a reaction tube, mix gently by inverting once and place tube in the Spec20 to obtain a "dark reading" with the spectrophotometer set at 580nm.  This is the time = zero seconds absorbance reading.  Take care not to shield the tube from the light with your hand.  Take absorbance readings every 15s (10s illumination + 5s reading) for 90s (7 readings).  Record absorbance readings and times in your lab notebook.  Repeat the experiment, i.e., do several 90s runs, until you obtain the desired reference rate of DCPIP reduction.  Then repeat the experiment, to see if you can reproduce your data consistently.

5.  Plotting the Data:  The reduction of DCPIP is linear until the curve begins to level off near the end of the reaction.  Since you are interested in the rate of DCPIP reduction, you are primarily concerned with the straight-line portion of the curve.  To facilitate your comparison of the different rates of reaction, plot your data as a scatter graph, and add a trendline (simple linear regression: [[Appendix E]]).  Include in your regression plots only data points that are in the linear portion of the curve.  Do not include data points that are clearly in the leveled-off portion of the curve.  Regression plots allow us to compare any two 90s experiments in a quantitative manner, using the slopes of the regression lines.