Jones Lab:Protocols:Sterilise seed

Sterilise Seed with chlorine gas

1) Place small number of seed (max 300/~5mm in eppendorf) and open lids

2) Place to desiccator

3) Add 3ml HCl (5M) to 50ml commercial bleach and quickly add desiccator lid THIS STEP IN FUME HOOD- CHLORINE GAS PRODUCED

4) After 3hrs, transfer to sterile hood

5) Leave for 20mins for residual gas to evaporate, then add ~500ul dH2O

6) Dispose of HCl/bleach mixture in sink in fume hood WITH EXCESS WATER (~5min of tap running)

7) Move seed to fridge/cold room for 3 days

8) Plate

Sterilise Seed with Ethanol

1) 5 min 70% ethanol

2) 10 min 100% ethanol

3) Water washes

4) Stratify 3 days 4°C

5) Plate seed