BISC110/S11: Series 3 Experiment 9 Hill Reaction: Difference between revisions

'''Running the Hill reaction''' (work in pairs)

'''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]<br>

'''Blanking the Spectrophotometer '''([[Appendix_B/F10 | 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. <br>

'''Varying the Reaction Rate:'''  The Hill reaction occurs at different rates depending on the amount of light supplied to the thylakoids.  You will measure the reaction rate by taking absorbance readings over a 90s period in 15s intervals.  We will use the drop in absorbance over time as a measure of the rate of the Hill reaction. <br>