User:Kalkao/notebook/Making Plaques

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  1. Obtain T-broth soft agar (0.7% agar)
  2. Place in the microwave for approximately 2 minutes and 45 seconds at 50% power
    • Make sure the cap to the bottle is unscrewed and loose so that the bottle does not explode
    • Place paper towels under the bottle to keep the microwave clean
  3. Place test tubes in the heat block set at level 5
  4. Place the soft agar in the 55*C incubator
  5. Obtain one 1.7mL centrifuge tube for every test tube in the heat block
  6. Add 250uL of overnight culture to each centrifuge tube
    • Use fireboy to sterilize each pipette before placing in culture
    • Flame each test tube opening before and after each use
  7. Obtain one pre-poured T-broth agar plate from the 4*C for every test tube in the heating block
  8. Obtain a lysate stock with phage
  9. Make several dilutions of the phage stock in T-broth to obtain a final dilution stock of approximately 1000 PFU/mL
  10. Obtain soft agar from incubator
  11. Pipette 3mL soft agar into each tube on the heat block
    • Sterilize the pipette before placing in soft agar container
    • Flame the test tube opening before and after each addition
  12. Pipette 2mL of the final diluted stock into a 2mL centrifuge tube
    • Sterilize the pipette before placing in the dilution stock
    • Sterilize the test tube before and after the pipette is inside
  13. Vortex the 2mL centrifuge tube
  14. Add phage from 2mL centrifuge tube to a tube with overnight culture
    • Vortex the phage stock before extracting lysate
    • Use a micropipette to obtain lysate (range from 10uL to 200uL to get between 100 to 300 phage per plate)
    • After adding the phage, vortex the mixture
  15. Add the mixture to a test tube from the heat block
    • Use a micropipette to suck up the whole mixture from the centrifuge tube
    • When adding the mixture to the soft agar, pipette up and down to mix contents
    • Flame the opening of the test tube before and after the addition
  16. Pour the phage/bacteria/soft agar mixture onto a plate
    • Swirl the plate gently to ensure that the whole plate is covered evenly
    • Do this quickly because the soft agar solidifies quickly
    • Touch the lip of the tube to the agar to make sure all of the mixture leaves the test tube
  17. Place empty test tube in bleach bin, and place the cap in the white metal bin next to the sink
  18. Repeat steps 13 through 17 for every plate
    • For blanks, use 250uL of distilled water to add to overnight culture tubes
  19. After agar has solidified, move the plates into the 37*C incubation room
    • Plaques should form in approximately 4 hours