Griffitts:Competition

From OpenWetWare

Revision as of 17:08, 20 February 2013 by Matthew B. Crook (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

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%.

Personal tools