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Revision as of 14:42, 8 July 2010 (view source) Jmason (Talk | contribs) ← Older edit		Revision as of 15:32, 8 July 2010 (view source) Jmason (Talk | contribs) m (→Solutions) Newer edit →
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-	==Solutions==	+	==Materials==
	*Sterile distilled water		*Sterile distilled water

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Revision as of 15:32, 8 July 2010

Materials

• Sterile distilled water
• 70% ethanol
• 50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old

Procedure

1. Transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
2. Spin down seed briefly in microfuge
3. Decant water, add 500 uL 70% EtOH, vortex, spin
4. Decant EtOH, rinse with sterile water, spin and decant
5. Add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant
6. Add 0.8 mL sterile water, vortex, spin, decant
7. Repeat Step 6 six times
8. Leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days

Notes

• Keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
• Multiple water washes are to remove all traces of bleach which also affects germination
• Do the procedure in a hood in the tissue culture room
• Store washed seed for stratification in refrigerator vegetable drawer in the prep room