Buffers for Chloroplast Isolation from Diatoms
Isolation Buffer ( 40 mL )
1 ) Mix :
20 ml 1 M sorbitol
400 μl 0.6 M Na2 - EDTA
200 μl 1 M MgCl
2400 μl 1 M KCl
40 μl 1 M MnCl
22 ml 1 M HEPES - KOH pH = 8.0
16.96 ml Sterile H2O .
2 ) Add bovine serum albumen ( BSA ) 1 % ( w / v ) just before use to subset of isolation buffer needed at the beginning of the procedure PEG - 6000 Solution .
Mix :
6 g PEG
10 ml water
Percoll Solution ( 20 mL Stock ) .
Mix :
19 ml Percoll
1 ml PEG – 6000 solution
0.2 g Ficoll
0.2 g BSA Gradient Mixture ( 10 mL Stock ) .
Mix :
250 μl 1 M HEPES - KOH pH = 8.0
1 ml 0.1 M EDTA
6.26 ml 1 M sorbitol
2.49 ml water 3X Bottom Layer ( 80 % Percoll ) .
Mix :
5.7 ml Percoll Solution
1.01 ml Gradient Mixture 3X Top Layer ( 40 % Percoll ) .
Mix :
5.04 ml Percoll Solution
6.96 ml Gradient Mixture 10M Ammonium Fluoride ( NH4F ) .
Mix :
3.7 g NH4F
10 ml H2O
