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Revision as of 14:14, 8 July 2010 (view source) Lgyoung (Talk | contribs) (New page: Seed Sterilization Solutions 1. Sterile distilled wate 2. 70% ethanol 3. 50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old [[P...) ← Older edit		Latest revision as of 19:36, 8 July 2010 (view source) Lgyoung (Talk | contribs)
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-	Seed Sterilization	+	==Materials==
		+	*Sterile distilled water
		+	*70% ethanol
		+	*50% bleach/1 drop [http://en.wikipedia.org/wiki/Tween_20 Tween] 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old
-	[[Solutions]]	+	==Procedure==
-	1. Sterile distilled wate	+	#Transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
-	2. 70% ethanol	+	#Spin down seed briefly in microfuge
-	3. 50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old	+	#Decant water, add 500 uL 70% EtOH, vortex, spin
		+	#Decant EtOH, rinse with sterile water, spin and decant
		+	#Add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant
		+	#Add 0.8 mL sterile water, vortex, spin, decant
		+	#Repeat Step 6 six times
		+	#Leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days
-	[[Procedure]]	+	==Notes==
-	1. transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water	+	*Keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
-	2. spin down seed briefly in microfuge	+	*Multiple water washes are to remove all traces of bleach which also affects germination
-	3. decant water, add 500 uL 70% EtOH, vortex, spin	+	*Do the procedure in a hood in the tissue culture room
-	4. decant EtOH, rinse with sterile water, spin and decant	+	*Store washed seed for stratification in refrigerator vegetable drawer in the prep room
-	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:	+	Courtesy of Mike Poling, Lab Technician: Horticulture & Landscape Architecture, Purdue University.
-	1. keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination	+
-	2. multiple water washes are to remove all traces of bleach which also affects germination	+
-	3. do the procedure in a hood in the tissue culture room	+
-	4. store washed seed for stratification in refrigerator vegetable drawer in the prep room	+

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Latest revision as of 19:36, 8 July 2010

Materials

• Sterile distilled water
• 70% ethanol
• 50% bleach/1 drop [http://en.wikipedia.org/wiki/Tween_20 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

Courtesy of Mike Poling, Lab Technician: Horticulture & Landscape Architecture, Purdue University.