<p> Lab 07 Light Reactions of Photosynthesis</p><p>Set up six stations (one on each bench), each with the following:</p><p>1. one spectrophotometer (test to see that it is working) 2. seven spectrophotometer tubes plus one small rack to hold these (stoppers to fit two of these tubes) 3. one 15 mL Falcon tube with about 10 mL 90% acetone 4. one 15 mL Falcon tube with about 5 mL chlorophyll extract (from spinach leaves) in 90% acetone. Should be a pale green color. 5. one halogen light source (test to see that it is working) 6. one cooler with crushed ice (check Inge's ice maker) 7. one stop watch 8. one 5 mL pipette with pipette filler 9. one 1000 uL pipettor with pipette tip box 10. one waste container for pipette tips 11. test tube rack 12. one 50 mL Falcon tube with 25 mL buffer (label: "Phosphate Buffer") 13. one 15 mL Falcon tube with 10 mL 2x10-4M DCPIP solution 14. one 15 mL Falcon tube with 10 mL distilled water 15. DCMU concentrations in 15 mL Falcon tubes (3 mL of each in a tube): a. 10-5 M (Sol'n A) b. 10-6 M (Sol'n B) c. 10-7 M (Sol'n C)</p><p>At the front bench or on a cart:</p><p>1. Fresh spinach leaves (for chloroplast preparation) 2. Blender 3. In a single large test tube, a very concentrated solution of chlorophyll extracted into acetone (should be dark green) 4. One slide projector 5. Assorted color filters 6. 250 mL Beakers for waste (label these): 1. acetone/chlorophyll extract 2. chloroplast suspension with DCPIP & DCMU 2 x 10-4 DCPIP Preparation</p><p>DCPIP = 2,6-dichloroindophenol (Sigma D-1878; Sodium Salt; FW = 290.1)</p><p>First prepare 100 mL of a 1x10-3 M solution (label as 10-3 M DCPIP): 290.1*(0.001)/10 = 0.0290 g in 100 mL distilled water</p><p>Then prepare 100 mL 2x10-4 M solution as follows (label as 2x10-4 M DCPIP): 20 mL of 10-3 M DCPIP in 80 mL distilled water</p><p>DCMU Preparion</p><p>DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea (FW = 233.1)</p><p>Need Several Concentrations (Be sure to label containers)</p><p>Stock Solution OO 1x10-3 M DCMU</p><p>100 mL final volume 233.1*(0.001)/10 = 0.0233 g in 100 mL distilled water</p><p>Sol'n A = 1x10-5 M DCMU (final volume 100 mL)</p><p>1.00 mL Stock Solutio'n OO in 99 mL distilled water</p><p>Sol'n B = 1x10-6 M DCMU (final volume 100 mL)</p><p>10.0 mL Sol'n A in 90 mL distilled water</p><p>Sol'n C = 1x10-7 M DCMU (final volume 100 mL)</p><p>10.0 mL Sol'n B in 90 mL distilled water pH 7.3 Phosphate Buffer</p><p>First make Stock Solutions</p><p>0.2 M Monobasic Stock</p><p>Combine 13.9 g sodium phosphate monobasic 500 mL dH20</p><p>0.2 M Dibasic Stock</p><p>Combine 53.65 g sodium phosphate dibasic heptahydrate (or 28.4 g of the anhydrous form) 1 L dH20</p><p>0.1 M pH 7.3 Phosphate Buffer</p><p>Combine 1200 ml dH2O and 138 mL of monobasic stock and 462 mL of dibasic stock to obtain pH = 7.3.</p><p>0.05 Phosphate Buffer with 0.4 M sucrose and 0.01 M KCl (2 L final volume)</p><p>Combine together the following ingredients:</p><p>1. 273.6 g sucrose 2. 1.491 g KCl 3. 1 L 0.1 M phosphate buffer (pH 7.3; see above) 4. add enough distilled water to achieve a final volume of 2 L.</p>