Experiment on the isolation of chloroplasts from Arabidopsis thaliana

Summary

This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.

Operation method

Isolation of chloroplasts from Arabidopsis thaliana

Materials and Instruments

Leaf tissue
Grinding Suspension Buffer
Beaker Polytron homogenizer

Move

1. Tissue homogenization

(1) Collect 10 g of leaf tissue into a 400 ml beaker.

(2) Add 200 ml of ice-cold grinding suspension buffer. Homogenize leaf tissue in 6 to 7 3-second pulses with a Polytron homogenizer in a 4°C room.

Grinding Resuspension Buffer:

10 mmol/L EDTA

50 mmol/L HEPES-KOH, pH 8.0

0.33 mol/L mannitol

0.5 g/L BSA ( fatty acid free)

5 mmol/L ascorbate (added before use)

(3) Homogenize by filtration through a mesh layer of mirabil (Calbiochem) and pour the filtrate into four 50 ml centrifuge tubes.

2. Centrifuge the homogenate at 8000 g (Sorvall HB-4 turntable, 7000 r/min) for one minute.

3. Carefully decant the supernatant. Resuspend the chloroplasts with a camel hair brush, using gentle movements.

4. Pipette 2 ml of 1x SH containing 35% Pecoll into a 15 ml centrifuge tube as a buffer layer: cover with resuspended chloroplasts. centrifuge at 13,000 g (Sorvall HB-4 turntable, 10,000 r/min) for 3 minutes.

5. Discard the supernatant, wash the precipitate twice with 15 ml of 1x SH, and resuspend with an appropriate volume of 1x SH.

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