"

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