Standard Operating Procedure
Title:
Department: Agronomy
Created by: Danielle Dykema, Kendra Meade
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Bill Beavis
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview:
This procedure describes seed surface sterilization for Arabidopsis thaliana. Sterilization is
completed with at 50% EtOH solution to remove any microbial contaminants from the seed
surfaces prior to plating. The protocol described here specifically addresses sterilization and
further procedures will address plating of the seeds and transfer of the seedlings to soil.
Equipment and reagents necessary:
Reagents:
25mL bleach
50mL dIH2O
500µL Tween-20
10mL of 100% ethanol
400µL of 50% EtOH
Equipment:
Lab coat
Safety glasses
Microcentrifuge tube rack
Timer
25 mL graduated cylinder
60mL syringe
~50-100ul of Arabidopsis seeds
sterile hood
4ºC refrigerator
Prepared Murashige and Skoog media plates
Permanent marker
1000µL micropipette
1mL Pipette
Disposables:
50mL Falcon tube - varies
20 µm filter tip
1.5mL microcentrifuge tube
pipette tips
surgical tape
Nitrile gloves
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Kemwipes
Procedure:
For Seed Sterilization solutions:
1. Turn on laminar flow hood four hours before use and wipe down with 75% EtOH solution
from media mixing and pouring
2. Sterilize equipment: clean pipette, tip box, tube rack, marker, timer, and gloves with 75%
EtOH solution
3. Label a 50mL Falcon tube “Seed Sterilization Solution” with a permanent marker
4. To the Falcon tube, add 25mL bleach, 25mL dIH2O and 500µL Tween-20 and mix tube
gently.
5. In the sterile hood, pour solution into a 60mL syringe with a 20 µm filter tip attached to it;
filter the solution into a new 50mL Falcon tube.
6. Label a second 50mL Falcon tube “50% Ethanol Solution” and add 10mL of 100% ethanol
and 10mL of sterile dIH2O using the 25mL graduated cylinder to the tube and mix gently.
Seed Sterilization process:
1. In a 1.5mL microcentrifuge tube, measure out desired amount of Arabidopsis seeds.
2. In a sterile hood, add 400µL of prepared 50% EtOH solution to tube using a 1mL pipette,
close and invert tube gently for 1 minute.
3. Remove solution from tube using a new pipette tip.
4. Add 400µL of Seed Sterilization to tube, close and invert tube gently for 10 minutes*
5. Remove solution from tube using a new pipette tip.
6. Rinse tube 3x with 400µL of sterile dIH2O, inverting tube gently 1 minute for each rinse,
remove solution from tube using a new pipette tip.
7. After 3rd rinse, take ~100µL of seed/dIH2O mixture and plate 10-15 seeds per plate.
8. Seal plate with surgical tape and place right side up in 4oC for 4 days and then place in
growth chamber.
*During the 10 minutes, remove plates from 4oC and place in hood to help reduce
condensation on plates
Personal Protective Equipment / Engineering Controls:
Lab coat, eye protection and hand protection must be selected as required by Section D of the EH&S
Laboratory Safety Manual. Check the box(es) next to the item(s) which are appropriate for your
procedure.
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
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Handling & Storage Precautions:
Handling: Avoid breathing dust, mist, or vapor. Keep container tightly closed. Avoid contact with
clothing and other combustible materials. Avoid ingestion and inhalation. Use with adequate
ventilation. Use only in a chemical fume hood. Discard contaminated shoes. Storage: Store in a
tightly closed container. Keep refrigerated. (Store below 4�C/39�F.)
Waste Disposal Procedures:
Dispose of in a manner consistent with federal, state, and local regulations.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Absorb spill with inert material (e.g. vermiculite, sand or earth), then place in suitable container.
Clean up spills immediately, observing precautions in the Protective Equipment section. Provide
ventilation.
Health & Safety Summary for Required Reagents:
Brief description of associated hazards
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 The above summary consists of guidelines for proper handling & disposal of
chemicals used in this procedure. You must read and understand the contents
of the entire MSDS(s) before starting this procedure.
References:
List references used in preparing this procedure.
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