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Extraction of Arabidopsis seeds

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 75C 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.

 Copyright 2005 @ Kansas Lipidomics Research Center , Kansas State University.
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 design by Prashanth Palakollu.

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Page last updated January 17, 2008