Griffitts:Competition
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Materials
- 1.5% bleach‡
- 75% EtOH
- LB-20% glycerol
- glass plate
- tweezers
- razor blades
- micropestles
- 1.7 mL microcentrifuge tubes
- Vortexer
- pipettes and tips
- LB broth
- sterile ddH2O
- Bunsen burner
- 100% EtOH
- spreader
- 50-mL Falcon tubes
- LB-Sm-Xgal plates
Procedure
- Label plates and tubes
- Divide plates into thirds
- Pour sterile water onto the glass plate
- Carefully remove a plant from the Turface and sever the roots from stem with a razor blade
- Remove residual rocks
- Place entire root system in a 50 mL falcon tube containing 5 mL LB broth
- Vortex for 30 seconds
- This constitutes the surface sample
- Remove the root system from the Falcon tube and place in a 1.5 mL microcentrifuge tube
- Add 700 μL of 75% EtOH
- Close lid, and invert 10 times
- Remove the EtOH with a p1000
- Be sure to remove residual EtOH from the cap as well
- Add 700 μL 1.5% bleach
- Invert 5 times
- Remove the bleach with a p1000
- Be sure to remove residual bleach from the cap as well
- Rinse 5 times with ddH2O
- Be sure to remove residual ddH2O from the cap each time
- Add 250 μL LB broth
- Thoroughly rush the root system with a micropestle
- Make serial dilutions of each sample
- For surface samples, make 10–1, 10–2, and 10–3 dilutions
- For crushed root samples, make 10–2, 10–3, and 10–4 dilutions
- Plate 10 μL of each dilution on an LB-Sm-Xgal plate
- Plate all three dilutions from each sample on different thirds of the same plate
- Use the corner of the spreader
- Incubate at 30°C for 2–3 days
- Add 200 μL LB-20% glycerol to what remains of the samples and freeze as backup in the –80°C freezer.
Notes
‡Clorox is 5.95% bleach, so dilute it 1:4 to get ~1.5%.