Experiments

Phage Propagation

  1. The phi EA 104 phage was ordered from ATCC. The protocol for propagation was followed as directed by the website.

    • Prepare actively growth culture of host strain in the recommended media. Allow to grow, shaking for 18-24hrs (overnight) at 26Cº.
    • Add 1mL of the recommended broth to the freeze-dried phage, and .5mL to a cry-vial.
    • Prewarm plates in incubator, overlay the solid plates with soft agar media (.5% agar) which has been inoculated with 1-2drops of the host culture.
    • Prepare dilutions of the resuspended phage by moving 100uL of the suspension into 900uL of media (1:10) and serial passage as needed to prepare additional dilutions.
    • Once the soft agar overlay has hardened, add dilutions of the page, dropwise, to the plates. A new plate should be prepared for every dilution of phage desired. Add the phage to the soft agar prior to pouring and hardening.
    • After 24hrs of growth, scrape off the soft agar and centrifuge at 1000rpm for 25min to pellet the cellular debris and the soft agar.
    • Save the supernatant and pass through a .22 filter and save the lysate at -20C.

    When plaques are formed and identified, 5ml of nutrient broth is added to each plate to do overnight growth once again. The next day, the soft agar and liquid is scraped into the 15ml tube, centrifuged at the conditions specified above in the ATCC protocol, and filtered to make the lysate.

    Fig 2

  2. Genomic Extraction

    Using the New England Biolabs High Molecular Weight DNA Extraction Kit, the phage DNA was attempted to undergo isolation.

  4. Centrifuge the culture at 12000g for 25min to pellet the cellular debris.
  5. Next, precipitate phages by incubating the filtered supernatant with 10% PEG and 1M NaCl at 4Cº to overnight.
  6. Precipitated phages were collected by centrifugation at 12000g for 10min. Remove the supernatant.
  7. Resuspend the phage in 50 mM Tris-HCl pH 7.5 buffer, 10 mM MgCl , and 100mM NaCl.
  8. The phage was lysed using 300uL of the HWM gDNA Tissue Lysis Buffer and 20uL of proteinase K for 45 minutes at 56ºC shaking at 300rpm
  9. 300uL protein separation solution was added and gently mixed. Centrifuge at 32000g for 10min.
  10. Transfer the resulting (top) aqueous layer to a new tube. Add two DNA capture beads.
  11. DNA precipitation was initiated by adding 550uL isopropanol. Slowly, gently, manually invert 25 times.
  12. Remove the supernatant by pipetting carefully to not disturb the DNA bound to beads.Wash 2x with 500uL gDNA wash buffer, removing the wash buffer in the same way as above.
  13. Pour beads into the provided container and pulse in a microcentrifuge to remove residual wash buffer.
  14. Add the DNA beads to 100uL of gDNA elution buffer II and incubate at 56ºC for 5minutes to allow the DNA to be released from the beads
  15. Transfer to a bead retainer to separate the beads from DNA by centrifugation at 12000g for 1min.

    16. After various iterations of the genomic extraction protocol ranging from trying to omit steps from the NEB protocol to fulfilling every step as listed, there was not sufficient DNA concentration to work with in planned experimental methods. Thus, we were not able to move forward with our cloning project and determined that the phage selected is not robust enough in a lab setting to be manipulated for a product that would be able to be used in orchards

Conclusion

Summarize the process and discuss any final thoughts.