Buffers for Chloroplast Isolation from Diatoms
Isolation Buffer (40 mL)
1) Mix:
20 ml 1 M sorbitol400 μl 0.6 M Na2-EDTA200 μl 1 M MgCl2400 μl 1 M KCl40 μl 1 M MnCl22 ml 1 M HEPES-KOH pH = 8.016.96 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 PEG10 ml water   Percoll Solution (20 mL Stock).
Mix:19 ml Percoll1 ml PEG – 6000 solution0.2 g Ficoll0.2 g BSA Gradient Mixture (10 mL Stock).
Mix:250 μl 1 M HEPES-KOH pH = 8.01 ml 0.1 M EDTA6.26 ml 1 M sorbitol2.49 ml water 3X Bottom Layer (80% Percoll).
Mix:5.7 ml Percoll Solution1.01 ml Gradient Mixture 3X Top Layer (40% Percoll).
Mix:5.04 ml Percoll Solution6.96 ml Gradient Mixture 10M Ammonium Fluoride (NH4F).
Mix:3.7 g NH4F10 ml H2O
