DPIP When chloroplasts are exposed to light, electrons reduce NADP + to NADPH
ELECTRON ACCEPTOR DPIP= substitute for NADP + DPIP ox = BLUE DPIP re = colorless
SPECTROPHOTOMETER Measures % light transmitted If DPIP is oxidized (blue) % transmittance will be low If electrons are excited and move to reduce DPIP(colorless) % transmittance will increase
SET UP LIGHT SINK Image modified from Holtzclaw & Holtzclaw Study Guide
Cover tube # 2 completely with foil Make a removable foil top
SET UP TUBES Add Phosphate buffer, distilled water, & DPIP to tubes as shown WAIT TO ADD CHLOROPLASTS!
SPECTROPHOTOMETER BLANK has all ingredients except DPIP Use Blank to calibrate machine Spectrophotometer is not digital Slow to respond Adjust dial and WAIT! BE PATIENT!
ZERO MACHINE Place BLANK in chamber and close lid Slowly turn dial on front until window reads 100% (Wait for lag time!) 100% with blank 0% with no tube/door closed
Add 3 drops of UNBOILED CHLOROPLASTS to tube #2 Put Parafilm on top and gently mix Remove from foil sleeve and immediately put sample in chamber, read % transmittance, and record in lab book Remove sample from chamber and return to foil sleeve, cover and place in test tube rack under light Start timer Take and record readings at 5, 10, & 15 min
REPEAT WITH EACH TUBE You can run multiple timers Just make sure you stagger time points so you are not taking readings of multiple tubes at same time Use cuvette #1 (blank) occasionally to check and adjust the spectrophotometer to 100% transmittance
Image modified from Holtzclaw & Holtzclaw Study Guide