Team:Purdue/Seed sterilization

From 2010.igem.org

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Seed Sterilization
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==Materials==
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*Sterile distilled water
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*70% ethanol
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*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
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[[Solutions]]
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==Procedure==
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1. Sterile distilled water
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#Transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
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#Spin down seed briefly in microfuge
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#Decant water, add 500 uL 70% EtOH, vortex, spin
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#Decant EtOH, rinse with sterile water, spin and decant
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#Add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant
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#Add 0.8 mL sterile water, vortex, spin, decant
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#Repeat Step 6 six times
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#Leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days
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2. 70% ethanol
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==Notes==
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3. 50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old
+
*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
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[[Procedure]]
+
Courtesy of Mike Poling, Lab Technician: Horticulture & Landscape Architecture, Purdue University.
-
 
+
-
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:]]
+
-
 
+
-
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
+

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.