Bleach Sterilization
1. Aliquot seeds into 1.5 mL microcentrifuge tube.
2. Prepare a 50% bleach with addition of 0.05% Tween
® 20 solution (see Note 1).
3. Starting from this step, perform all the steps in sterile conditions in a laminar flow hood. Add 500 μL 50% bleach solution
to the microcentrifuge tube containing the seeds.
4. Allow the seeds to soak for 10 min. To aid in sterilization, tap
the bottom of the tube to suspend the seeds in the bleach
solution.
5. Rinse the seeds with 500 μL of sterile distilled water seven times
(see Note 2).
6. Seeds are ready to be placed on desired solid media.
Chlorine Gas Sterilization
1. Aliquot seeds to be sterilized into a 1.5 mL
microcentrifuge tube.
2. Prepare materials for chlorine gas sterilization (see Note 3).
3. Calculate the amount of bleach and HCl required for sterilization (see Notes 4 and 5).
4. Starting from this step, perform all the steps in sterile conditions in a fume hood. Open the caps of all the microcentrifuge
tubes in the seed rack and place the rack of seeds in sterilization
container (see Note 6).
5. Place a beaker inside the container and add the correct amount
of bleach (see Note 7).
6. Carefully add the correct amount of HCl to the beaker containing the bleach.
7. Close the sterilization container and seal it with Parafilm
®
immediately (see Note 8).
8. Allow the container to sit in the fume hood for 1 h.
9. Remove the Parafilm
® from the container and carefully open
the lid on one corner. Allow the container to vent for 3 h to
complete the reaction and eliminate the chlorine gas.
10. Close the caps of all the microcentrifuge tubes in the seed rack.
Remove the seed rack and place it in a laminar flow hood. Seeds
can be stored in dry conditions at 20–30% relative humidity
(RH) until ready to place on solid media.
11. Neutralize the chlorine gas reaction with sodium bicarbonate
(NaHCO 3 ) for disposal (see Note 9).
12. Seeds are ready to be placed on desired solid media.
Handling Arabidopsis Plants
7
1. Aliquot seeds into 1.5 mL microcentrifuge tube.
2. Prepare a 50% bleach with addition of 0.05% Tween
® 20 solution (see Note 1).
3. Starting from this step, perform all the steps in sterile conditions in a laminar flow hood. Add 500 μL 50% bleach solution
to the microcentrifuge tube containing the seeds.
4. Allow the seeds to soak for 10 min. To aid in sterilization, tap
the bottom of the tube to suspend the seeds in the bleach
solution.
5. Rinse the seeds with 500 μL of sterile distilled water seven times
(see Note 2).
6. Seeds are ready to be placed on desired solid media.
Chlorine Gas Sterilization
1. Aliquot seeds to be sterilized into a 1.5 mL
microcentrifuge tube.
2. Prepare materials for chlorine gas sterilization (see Note 3).
3. Calculate the amount of bleach and HCl required for sterilization (see Notes 4 and 5).
4. Starting from this step, perform all the steps in sterile conditions in a fume hood. Open the caps of all the microcentrifuge
tubes in the seed rack and place the rack of seeds in sterilization
container (see Note 6).
5. Place a beaker inside the container and add the correct amount
of bleach (see Note 7).
6. Carefully add the correct amount of HCl to the beaker containing the bleach.
7. Close the sterilization container and seal it with Parafilm
®
immediately (see Note 8).
8. Allow the container to sit in the fume hood for 1 h.
9. Remove the Parafilm
® from the container and carefully open
the lid on one corner. Allow the container to vent for 3 h to
complete the reaction and eliminate the chlorine gas.
10. Close the caps of all the microcentrifuge tubes in the seed rack.
Remove the seed rack and place it in a laminar flow hood. Seeds
can be stored in dry conditions at 20–30% relative humidity
(RH) until ready to place on solid media.
11. Neutralize the chlorine gas reaction with sodium bicarbonate
(NaHCO 3 ) for disposal (see Note 9).
12. Seeds are ready to be placed on desired solid media.
Handling Arabidopsis Plants
7