Table for recordingDCIP reduction A₆₀₀ versus time of light exposure.

18 Sept 00, 20 Nov 01

Copy this table into your notebook prior to starting the chloroplast reduction experiment. Every blank square gets a reading.

vol.
chloroplasts light
cond
Time of exposure in minutes

0 1 2 3 4 5 6 7 8 9 10 15

0 dark x x x x x x x x x x

0 light x x x x x x x x

10uL dark x x x x x x x x x x

10uL light

50uL dark x x x x x x x x x x

50 uL light

CHLOROPLAST REDUCTION OF INDOPHENOL

David B. Fankhauser, Ph.D.

18 November 1992, rvsd 23 Nov 93, 20 Sept 94, 21 Nov 95, 20 Sept 96, 26 Nov 96, 20 Sept 99, 18 Sept 00

From DBF Notebook, C.C. III, p. 84 & CC IX, p. 107

See previous protocol for the preparation of the purified chloroplasts. Continue to keep them ice cold until the moment you add them to the prepared tubes. Teams of two.

Supplies:

purified spinach chloroplasts, ice cold
0.1 M PO₄ buffer, pH 6.5 (pH 7.0 OK?)
2.5 x 10^-4 M 2,6 dichlorophenolindophenol
(36.3 mg DCIP/500 mL, A₆₀₀ 3.0)
displacement pipets
six unblemished 13x100 mm test tubes
100 watt light 1/ two teams
cuvette for water blank 1/team
37C incubator (can exclude light)

Set up hot block next to spectrophotometer:
hot block, 37C 1/team
spectrophotometer 1/team

1. Set up apparatus:
a: 37C hot block (for 13x100 mm tubes) nearby and warmed up.
b: 37C incubator warmed up.
c: spectrophotometer on the same desk as the light exposure apparatus.
d: 100 watt bulb with reflector 25 cm from open test tube rack.

Construct a data table in your notebook to accept data (previous page in handouts).

2. Select and label seven very clean, unblemished cuvettes (B for blank + six tubes).

Prepare DCIP reaction mix which should have an A₆₀₀ of 0.400 to 0.600.

For five teams: 60 mL 0.1 M PO₄, pH 6.5 buffer
60 mL 0.5 M sucrose
40 mL 2.5 x 10^-4 M DCIP

4. Dispense 4 mL of the reaction mix to each of your tubes with repeater pipetter.
5. Add all of the following ingredients except for the chloroplasts:

tube	mL Reaction Mix	µL Chloroplast suspension	Incubation conditions	tube	A₆₀₀
1	4.00	0	dark	1
2	4.00	0	light	2
3	4.00	10	dark	3
4	4.00	10	light	4
5	4.00	50	dark	5
6	4.00	50	light	6

6. Prewarm prepared tubes (lacking chloroplasts) in 37C hot block for 2 min.
7. Add diluted chloroplasts to tubes 3 and 5. Mix and read the A₆₀₀ of 1, 3, and 5 against a water blank (= T₀ for dark tubes). Place in 37C incubator, keep light excluded.
8. Add indicated volume of chloroplasts to tubes 2, 4 and 6. Mix and read A₆₀₀ of these tubes as in step 4 ( = T₀). Place 25 cm from a 100 watt bulb (set up next to the spectrophotometer).

9. Read A₆₀₀ of 4 and 6 every minute for 10 minutes, read tube 2 only at 0, 5, and 10 min. (Keep tubes clean, read in consistent configuration.)
10. Read A₆₀₀ of tubes which were kept in the dark again at 15 minutes.
11. Plot the absorbency of each tube versus time.

Discuss the significance of the differences between the various six curves.

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