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Team:Purdue/Seed sterilization
From 2010.igem.org
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==Solutions== | ==Solutions== | ||
| - | + | *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== | ==Procedure== | ||
| - | # | + | #Transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water |
| - | # | + | #Spin down seed briefly in microfuge |
| - | # | + | #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 |
==Notes== | ==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 | |
Revision as of 14:42, 8 July 2010
Solutions
- 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
- Transfer a small scoop of seed to an eppendorf tube; soak seed for 30 minutes in distelled water
- Spin down seed briefly in microfuge
- 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
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
