Griffitts:Competition

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

1. Label plates and tubes
   • Divide plates into thirds
2. Pour sterile water onto the glass plate
3. Carefully remove a plant from the Turface and sever the roots from stem with a razor blade
4. Remove residual rocks
5. Place entire root system in a 50 mL falcon tube containing 5 mL LB broth
6. Vortex for 30 seconds
   • This constitutes the surface sample
7. Remove the root system from the Falcon tube and place in a 1.5 mL microcentrifuge tube
8. Add 700 μL of 75% EtOH
9. Close lid, and invert 10 times
10. Remove the EtOH with a p1000
    • Be sure to remove residual EtOH from the cap as well
11. Add 700 μL 1.5% bleach
12. Invert 5 times
13. Remove the bleach with a p1000
14. Be sure to remove residual bleach from the cap as well
15. Rinse 5 times with ddH₂O
16. Be sure to remove residual ddH₂O from the cap each time
17. Add 250 μL LB broth
18. Thoroughly rush the root system with a micropestle
19. 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
20. 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
21. Incubate at 30°C for 2–3 days
22. 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%.