Team:Purdue/Seed sterilization

From 2010.igem.org

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==Solutions==
==Solutions==
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#Sterile distilled water
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*Sterile distilled water
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#70% ethanol
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*70% ethanol
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#50% bleach/1 drop Tween 20: prepare 40 ml in a Falcon tube, use bleach that is less than two months old
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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
==Procedure==
==Procedure==
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#transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
+
#Transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
-
#spin down seed briefly in microfuge
+
#Spin down seed briefly in microfuge
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#decant water, add 500 uL 70% EtOH, vortex, spin
+
#Decant water, add 500 uL 70% EtOH, vortex, spin
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#decant EtOH, rinse with sterile water, spin and decant
+
#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
+
#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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#Add 0.8 mL sterile water, vortex, spin, decant
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#repeat Step 6 six times
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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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#Leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days
==Notes==
==Notes==
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#keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
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*Keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination
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#multiple water washes are to remove all traces of bleach which also affects germination
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*Multiple water washes are to remove all traces of bleach which also affects germination
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#do the procedure in a hood in the tissue culture room
+
*Do the procedure in a hood in the tissue culture room
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#store washed seed for stratification in refrigerator vegetable drawer in the prep room
+
*Store washed seed for stratification in refrigerator vegetable drawer in the prep room

Revision as of 14:42, 8 July 2010

Solutions

  • 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