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Extraction of Arabidopsis seeds
NOTES:
Use glass tubes with Teflon-lined screw caps. Use a Dounce-type
homogenizer – ground glass to ground glass. Use glass Pasteur
pipets for transferring seeds/solvent.
Volumes
may be doubled for larger samples or ease of homogenizing.
1)
Weigh seeds. Lyophilizing first may be best for consistent dry
weight.
2)
Drop seeds into 1.0 ml 75ºC isopropanol with 0.01% BHT. Heat 15
minutes. Let cool. (Hot isopropanol inactivates phospholipase
enzymes.)
3)
Transfer to homogenizer; homogenize thoroughly. Transfer to 17 ml
glass tube for extraction. Rinse homogenizer with 1.0 ml chloroform
and 1.0 ml methanol to recover all the seed parts/lipids; add to
isopropanol/seed mix in tube. Add 0.8 ml water to solvent/seed
mix. Shake well – should be one phase.
4) Add
1.0 ml chloroform and 1.0 ml water. Shake well. Centrifuge to
split phases.
5)
Remove lower layer (chloroform and lipids). SAVE to clean glass
tube.
6) Add
1.0 ml chloroform, shake, and centrifuge. SAVE this lower layer in
same tube as first.
7) Add
1.0 ml chloroform, shake, and centrifuge. SAVE this lower layer in
same tube as above.
8) Add
small amount (0.5 ml) 1 M KCl to chloroform/lipids. Shake well;
centrifuge. Remove thin water layer on TOP and discard.
9) Add
small amount (1.0 ml) water to chloroform/lipids. Shake well;
centrifuge. Remove thin water layer on TOP and discard. (This
backwash removes any proteins and carbohydrates that may have been
carried through the extraction.)
10)
Dry to small volume. Transfer to 2.0 ml vial with Teflon-lined
screw cap, dry completely, fill vial with nitrogen gas, and ship
overnight on dry ice. Please contact Mary Roth at
mrroth@ksu.edu before shipping. |
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