Rationale: Now that I have enough lysate to run DNA extraction I can proceed with doing so and now I can view my TEM that I prepared a couple weeks ago.

Procedure:

Wednesday:

1. Pipetted 10mL of lysate into a 50mL conical vial.
2. Added 40µL of Nuclease Mix and gently shook the vial for about a minute.
3. Added 4mL of Phage Precipitant Solution and gently shook once again.
4. Incubated at 37°C for 30 minutes then took out of incubator and left at room temperature for 40 minutes.
5. Centrifuged at 10,000g for 20 minutes.
6. Decanted supernatant and stored pellet in freezer overnight.
7. Since I already prepped the TEM last week I just had to go in and view my phage under  the microscope.

Thursday:

1. Added 500µL of sterile water to pellet and re-suspended by pipetting up and down.
2. Added 2mL of resin to re-suspended pellet and pipetted up and down to stir.
3. Split up the solution into two microcentrifuge tubes and spun at 12.5g for 3 minutes.
4. Decanted the supernatant and added 1mL of 80% isopropanol to each tube. Mixed by flicking tube and gently swirling.
5. Spun at 12.5g for 3 minutes.
6. Repeated steps 4 and 5.
7. Decanted supernatant and added 1mL of 80% isopropanol. Re-suspended pellet.
8. Poured each tube into separate vacuum filters.
9. Added each column to a new microcentrifuge tube and spun at 12000g for 5 minutes.
10. Took column out and placed into another set of new microcentrifuge tubes. Added 100µL of elution buffer to column and let sit for a minute.
11. Spun at 12000g for 1 minute then placed the solution into one microcentrifuge tube. Stored at -20°C overnight.

Observations:

This image was produced using TEM. My phage has a tail length of 100nm.

Conclusions and Next Steps:
Now I need to find out how much nucleic acid is in my sample using the nanodrop machine. From there I will run PCR and gel electrophoresis to find out what cluster my phage is.