November 13

Lab Day 25: Flooding, Serial Dilutions, Web/Spot Test

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Rationale

Current titer calculations from 10-2 plate was 106. The 10-1 and 10-2  plates from previous lab day will be flooded and filtered out. A spot test will be performed using 50 µL of 100, 10-1, 10-2, 10-3 ,10-4 , and 10-5  of the new lysate with a goal to receive a webbed plate and get a high titer.

Detailed Procedure

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

Observations/Results/Next Step

Control plate from previous lab day had no contamination and size of plaques all seem to remain the same size. Plate seeme nearly fully webbed, it was enough to flood the plate. For the new plates, air bubbles were formed. The spot test for 10-1 and 10-2 collided with each other, so it might be hard to calculate the titer, but hopefully a high titer can be calculated for the rest of the spotted dilutions by next lab day.

10^-2 plate calculated a 10^6 titer

newly filtered lysate after flooding

10^0 — 10^-5 dilutions were made

results from lab day 24

spot test 11/13/18


Posted November 13, 2018 by soo-un_jeong1 in category Soo-Un Jeong

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