The resulting combination
dried under N2 at 40 C. The pellet was re-extracted for a third time
with a 41 mL solution prepared by mixing together 36 mL of 2:1 (v/
v) chloroform:methanol and 5 mL of 0.5 M KCl/0.8 M H3PO4,
followed by sonication for 2 min on ice. After vigorously mixing
the pellet with this solution for 2 min using a vortex, the mixture
centrifuged to create a phase separation, the lower lipid phase
removed, and then added to the dried mixture of first and second
extractions. The combination of the 3 extractions was dried under
N2 at 40 C.