Technical information
Centrifuge Standard Operating Procedure 23
10 x Import Master Mix
(50 mM HEPES, 330 mM Sorbitol, 400 mM KOAc, 50 mM MgOAc)
For 25 mL:
0.298 g HEPES
1.503 g sorbitol
0.981 g KOAc
0.268 g MgOAc
Adjust pH to 8.0 using KOH
Stored as 1 mL aliquots @ -20°C
Part I: Chloroplast Isolation
1. Prepare equipment;
a. thaw Percoll
b. cool down centrifuge (put in JLA 10.5 rotor and spin with at least
two water blanks at 1,000 x g for 5 min; rotor stored in cold room)
c. put 500 ml centrifuge bottle on ice (w/ funnel and Miracloth)
2. Prepare 1x Grinding Buffer:
a. To 125 ml of 2x GB add:
i. 4.95 g ascorbic acid
ii. 0.625 g BSA
b. Add milli-Q to a volume of ~225 mL and adjust pH to 7.5 using KOH
c. Bring volume up to 250 ml with milli-Q water
d. KEEP ON ICE
3. Prepare 2 Percoll step gradients (in 50 ml centrifuge tubes): 7 ml of 85%
Percoll on bottom, 8 mL of 35% Percoll on top.
4. Harvest green tissue by shaving off Arabidopsis tissue from the surface of
the plates using a single-sided razor into a 600 mL plastic beaker. Avoid
getting agar in the beaker.
5. Add ~200 mL of ice cold 1x grinding buffer to harvested tissue and
homogenize using PowerGen homogenizer (@ setting 3, ~15 sec).
6. Filter homogenate through 2 layers of Miracloth into a pre-chilled 500 ml
centrifuge bottle.
7. Centrifuge for 8 min, 1,000 x g, 4C (JLA 10.5 rotor) and decant SN (down
drain).
8. GENTLY re-suspend pellet in ~8 ml of fresh, ice-cold, 1x Grinding Buffer.
9. Divide chloroplast suspension evenly between 2 Percoll step gradients using
10 mL pipette.
10.Centrifuge gradients @ 7,700 x g, 15 min, and 4C in swinging bucket rotor
(JS13.1, in cold room) with SLOW acceleration/deceleration.
11.Aspirate top layer of broken chloroplasts and buffer (waste).
12.Collect lower band of intact chloroplasts using a Pasteur pipette and dilute
in ~50 ml of HS buffer (put ~20 ml of HS in a centrifuge tube, collect C.P.s,