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Team:Purdue/Seed sterilization
From 2010.igem.org
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1. Sterile distilled water | 1. Sterile distilled water | ||
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2. 70% ethanol | 2. 70% ethanol | ||
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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 | 3. 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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1. transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water | 1. transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water | ||
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2. spin down seed briefly in microfuge | 2. spin down seed briefly in microfuge | ||
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3. decant water, add 500 uL 70% EtOH, vortex, spin | 3. decant water, add 500 uL 70% EtOH, vortex, spin | ||
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4. decant EtOH, rinse with sterile water, spin and decant | 4. decant EtOH, rinse with sterile water, spin and decant | ||
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5. add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant | 5. add 0.8 mL 50% bleach, vortex for 8-10 min, spin and decant | ||
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6. add 0.8 mL sterile water, vortex, spin, decant | 6. add 0.8 mL sterile water, vortex, spin, decant | ||
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7. repeat Step 6 six times | 7. repeat Step 6 six times | ||
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8. leave seed in 0.5 mL of 0.12% sterile agar; allow to stratify at 4 degrees for at least 2 days | 8. 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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1. keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination | 1. keep the ethanol step as brief as possible-prolonged exposure to EtOH decreases germination | ||
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2. multiple water washes are to remove all traces of bleach which also affects germination | 2. multiple water washes are to remove all traces of bleach which also affects germination | ||
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3. do the procedure in a hood in the tissue culture room | 3. do the procedure in a hood in the tissue culture room | ||
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4. store washed seed for stratification in refrigerator vegetable drawer in the prep room | 4. store washed seed for stratification in refrigerator vegetable drawer in the prep room | ||
Revision as of 14:26, 8 July 2010
Seed Sterilization
1. Sterile distilled water
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
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
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
