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From 2010.igem.org

Seed Sterilization

Solutions

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

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:

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