November 28

Lab Day 30: Flooding, Serial Dilution, Spot Titer

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Rationale

5 microliters and 203 microliters plates were flooded and had serial dilutions performed up to 10^-12. A spot titer was performed to prevent making 13 plaque assay plates.

Detailed Procedure

  1. Add 8 mL of PB to  203 microliters  plate from previous lab day.
  2. Add 5 mL of PB to  5 microliters  plate from previous lab day.
  3. The plates was shaken on an incubator for one hour.
  4. Filtered lysate from flooded plate in two separate 50 mL 0.22 µm filtered conical vial. Labeled “SJ 11/28/18 FS 2 lysate 100” and “SJ 11/28/18 FS 3 lysate 100.”
  5. 10 µL of “SJ 11/28/18 FS 2 lysate 100” was added with 90 µL of phage buffer to create 10-1 dilution which was vortexed.
  6. 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
  7. 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
  8. 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
  9. 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
  10. 10 µL of 10-5 was added with 90 µL of phage buffer to create 10-6 dilution which was vortexed.
  11. 10 µL of 10-6 was added with 90 µL of phage buffer to create 10-7 dilution which was vortexed.
  12. 10 µL of 10-7 was added with 90 µL of phage buffer to create 10-8 dilution which was vortexed.
  13. 10 µL of 10-8 was added with 90 µL of phage buffer to create 10-9 dilution which was vortexed.
  14. 10 µL of 10-9 was added with 90 µL of phage buffer to create 10-10 dilution which was vortexed.
  15. 10 µL of 10-10 was added with 90 µL of phage buffer to create 10-11 dilution which was vortexed.
  16. 10 µL of 10-11 was added with 90 µL of phage buffer to create 10-12 dilution which was vortexed.
  17. 4 mL of LB Broth, 45 µL of CaCl2, and 5 mL of 2X TA were combined into a 15 mL vial.
  18. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/28/18.”
  19. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SJ Spot 11/28/18” which was sectioned off into 12 parts
  20. Waited 15 mins for both plates to solidify.
  21. Micropipetted 10 µL of 100, 10-1, 10-2, 10-3 ,10-4 , 10-5  , 10-6  , 10-7  , 10-8  , 10-9  , 10-10  , 10-11   , 10-12 in according sections of the spot test plate.
  22. Rested into incubator

Conclusion/Next Steps

203 microliter plate was completely lysed, which gives hope that a high titer will be achieved by next lab day. 5 microliter plate was nearly fully webbed, but not enough, so only 5 mL of PB was poured onto the plate. Each plate, after an hour, were filtered out in separate vials. Only the 203 microliter plate was used for serial dilutions because the plate was completely lysed. By next lab day, a new titer will be calculated and hope to achieve a high titer by then.

results from lab day 29 (below)

10^-1 through 10^-12 spot titer plate


Posted November 28, 2018 by soo-un_jeong1 in category Soo-Un Jeong

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