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Materials and Methods .
• Six 13 x 100 mm (10 ml) Test Tubes.
• Aluminum Foil (6 in.).
• 5 or 10 ml Pipet.
• Spectrophotometer.
• Test Tube Rack.
• Beaker of Distilled Water.
• Beaker of Student Chloroplast Dilution Buffer.
• DCIP dye .
• Sarkosyl.
• Flood Lights or Lamps.
• Paper Towels.
• Clock.
• Marker.
Experimental Procedures.
1. Label six 13 x 100 mm (10 ml) test tubes 1-6 at their tops.
2. Wrap tube 5 in foil, including top with the foil.
3. Using a 5 or 10 ml pipet, add 2.5 ml of Student Chloroplasts Dilution Buffer to each of the six test tubes.
4. The following chart (Table 1) describes the pipeting of reagents to each of the tubes. Use a designated pipet for each reagent. For example, use the same pipet for only the chloroplasts in a different pipet only for the DCIP dye, etc.
Mix the tubes thoroughly since the dilution buffer is denser than the other reagents. Pipet the sarkosyl in Tube 6 after ALL other pipetings have been completed. If there is an inadvertent cross contamination of the sarkosyl into the other tubes, it will ruin the experiment. Use 1 ml pipets for volumes of 1 ml or less.
5. Bring the tubes to the spectrophotometer. The instrument should be set at 605 nm. If you are using a Spectronic 20, zero the instrument as follows:.
WITHOUT a tube in the instrument, with the cover to the sample chamber CLOSED, adjust the instrument to read 0% transmittance by adjusting the left knob (power switch/ zero control). .
Open the sample chamber and put in Tube 1. Close chamber and adjust the instrument to read 100% transmittance with the right knob (transmittance absorbance control). .
6. Obtain % transmittance values for Tubes 3, 4, and 6. Record your results in the table under 0 time. Quickly remove the foil from Tube 5 and place it in the instrument. Record the reading and after removing the tube quickly wrap it back up in the foil.
