Technical information

Centrifuge Standard Operating Procedure 22
APPENDIX 1: ARABIDOPSIS CHLOROPLAST ISOLATION AND
CHLOROPHYLL DETERMINATION
This procedure is based on a method used by the M. Smith lab, Oct 2007.
Required Buffers
1M HEPES-KOH
-add 119.15g HEPES to ~300 mL of milli-Q H
2
0
-stir until dissolved; add milli-Q to 500 mL
-Adjust pH to 7.5 using 5M KOH
-Autoclave @ L30
HEPES SORBITOL Buffer (HS Buffer, 200 mL)
10mL 1M HEPES-KOH, pH 7.5 (50mM final)
12.02g Sorbitol (330mM final)
-adjust pH to 7.5 w/ KOH, volume up to 200mL with milli-Q
-store at 4C
2 x Grinding buffer (GB, 300 mL)
30mL 1M HEPES-KOH, pH 7.5 (100mM final)
36.07g Sorbitol (660mM final)
2.4 mL 0.5M EDTA (4mM final)
300μl 2M MgCl
2
(2mM final)
600μl 1M MnCl
2
(2mM final)
85% Percoll (50 mL)
42.5mL Percoll (85% final)
2.5mL 1M HEPES-KOH, pH 7.5 (50 mM final)
3.01g Sorbitol (330 mM final)
50μl 2M MgCl
2
(2mM final)
400μl 0.5M EDTA (4mM final)
0.1g BSA
0.5 g Ascorbic acid (50 mM final)
-Adjust pH to 7.5 with KOH; add milli-Q up to 50mL
-store at -20C
40% Percoll (50 mL)
20 mL Percoll (40% final)
25 mL 2x GB
0.5g Ascorbic acid (50mM final)
-Adjust pH to 7.5 with dilute HCl
-Add milli-Q up to 50 mL
-store at -20C