Arabidopsis Seed Sterilization and Plating

Procedure for seed sterilization:

1. Label microcentrifuge tubes.

2. Place seeds into microcentrifuge tubes.

3. Add 500l of 95% ethanol to each tube and incubate for 5 minutes. (Be sure not to wipe off your label with the ethanol.)

4. Remove ethanol.

5. Add 500l of freshly made bleach solution to each tube and incubate for 5 minutes.

Bleach solution*: 20% household bleach 0.1% Tween 20

6. Add 1ml of sterile dH2O, when seeds have settled, remove the diluted bleach solution.

7. Rinse seeds with 1ml sterile dH2O.

8. Remove water and repeat washing step twice more.

o 9. Add 1ml sterile dH2O, cap the microcentrifuge tubes and store seeds for ~48hrs at 4 C.

*1ml of bleach solution is made with: 0.2ml bleach and 0.001ml Tween 20.

Procedure for making MS plates and top agar:

Recipe for MS plates Recipe for top agar 4.3g MS salts 0.43g MS salts 10g sucrose 1g sucrose bring up volume to 900ml with dH2O bring up volume to 90ml with dH2O pH to 5.7-5.8 with 1M NaOH pH to 5.7-5.8 with 1M NaOH add 8g agar add 0.8g agar bring up volume to 1L with dH2O bring up volume to 100ml with dH2O autoclave for 20min autoclave for 20min

Procedure for plating:

1. Wipe down hood with 95% ethanol. 2. Top agar needs to be warm (melted, but not too hot as to kill the seeds).

3. Remove water from imbibed seeds.

4. Add 500l warm top agar to seeds. You will need to cut the tip of the pipette tip.

5. Carefully deposit the seeds onto the MS plate in a line. Do not work too slowly, as the agar will solidify in the tip.

6. Once the top agar has solidified on your plate, parafilm the plates shut and grow the plates vertically in a growth chamber.