PROCEDURES

CHLOROPLAST ISOLATION
The following procedure will provide enough chloroplast suspension for 4
groups:
1) Weigh out 40 g of fresh spinach (Spinacia oleracea) leaves
from which the major veins have been removed.
2) Tear the leaves into small pieces and place them in a blender
with 200 mls of cold isolation buffer (= 0.3 M sorbitol; 0.1 M
Tris-Cl, pH 7.8; 5 mM MgCl2; 10 mM NaCl).
3) Blend the mixture at low speed for 10 seconds. Use a glass rod
to push any tissue pieces down the sides of the jar into the
solution, and blend for 10 more seconds.
4) Filter the homogenate (called a tissue 'brei') through 8 layers of
cheesecloth into a beaker. Add approximately 40 ml of filtrate
to each of four 40 ml centrifuge tubes, making sure they are
balanced (+ 0.1 g).
5) Centrifuge at 4oC for 5 minutes at 1000 x g .
6) Decant and discard the supernatant from all 4 tubes. Add 0.5
mls of cold isolation buffer each tube and gently resuspend the
chloroplasts with a paint brush.
7) Store the centrifuge tube on ice, away from bright light.