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1) Pipet to 1 crushed grain: 500 µl (55 % (v/v) isopropanol / Bidest (+ 0.1 % DTT for barley only).
2) Vortex for 15 sec.
3) 15 min in ultrasonic tray at 40°C.
4) Vortex for 15 sec.
5) Centrifuge for 5 min 13,000 rpm.
6) Carefully pipet solutions into new tubes:
7) Remove alcohol for 2 - 3 hours in a SpeedVac under warming the rotor (solutions become turbid).
Caution! Remove isopropanol only: remaining aqueous volume is 50-100 µl.
8) Pipet out and discard the entire supernatant.
9) Add 0.6 g urea to 9 ml sample buffer (1 mol/l) + 1 ml Chloroethanol (10%, buffer becomes turbid), and add to the pellets: Barley-malt: 140 µl (+0.1% DTT), Wheat-malt 170 µl.
10) Vortex for 15 sec to dissolve the pellet to an emulsion.
11) 15 min ultrasonic tray at 40°C.
12) Vortex for 15 sec.
13) Sortly centrifuge the samples (1 min 1600 rpm).
See also the sequence of pictures: sortextkornextract.pdf
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