November 30

Lab Day 32: Flooding. Serial Dilutions, Spot Titer

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Rationale

10 microliter plate was flooded and had serial dilutions performed up to 10^-8. A spot titer was performed to calculate titer by Monday.

Detailed Procedure

  1. Add 5 mL of PB to  10 microliters  plate from previous lab day.
  2. The plates was shaken on an incubator for one hour.
  3. Filtered lysate from flooded plate in two separate 50 mL 0.22 µm filtered conical vial.
  4. 10 µL of 100 was added with 90 µL of phage buffer to create 10-1 dilution which was vortexed.
  5. 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
  6. 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
  7. 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
  8. 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
  9. 10 µL of 10-5 was added with 90 µL of phage buffer to create 10-6 dilution which was vortexed.
  10. 10 µL of 10-6 was added with 90 µL of phage buffer to create 10-7 dilution which was vortexed.
  11. 10 µL of 10-7 was added with 90 µL of phage buffer to create 10-8 dilution which was vortexed.
  12. 4 mL of LB Broth, 45 µL of CaCl2, and 5 mL of 2X TA were combined into a 15 mL vial.
  13. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/30/18.”
  14. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SJ Spot 11/30/18” which was sectioned off into 8 parts
  15. Waited 15 mins for both plates to solidify.
  16. Micropipetted 10 µL of 100, 10-1, 10-2, 10-3 ,10-4 , 10-5  , 10-6  , 10-7  , 10-8  in according sections of the spot test plate.
  17. Rested into incubator

Conclusion

10 microliter plate was completely lysed, which gives hope that a high titer will be achieved by Monday. Only 5 mL of PB was on the plate since the graduated pipette didn’t have up to 10 mL. By next lab day, a new titer will be calculated and hope to achieve a high titer by then.


Posted November 30, 2018 by soo-un_jeong1 in category Soo-Un Jeong

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