Rationale:

Continued with DNA Extraction and made serial dilutions to perform a spot titer test to calculate the titer of our plate. 

Procedure: 

1. The pellet was obtained from the freezer and 500 uL of sterile water was added. 
2. The solution was flicked, and the solution was evenly mixed using a pipette as well.  
3. Resin was obtained and 2 mL were added into solution containing the pellet.  
4. Pipetted the solution to mix thoroughly.  
5. Two microcentrifuge tubes were obtained, and 1.5 mL of solution was added to each.  
6. Both tubes were centrifuged at 12.5 xg for 3 minutes.  
7. Using a bulb pipette, the supernatant was removed, without touching the pellet.  
8. 1 mL of isopropanol was added to each microcentrifuge tube and pipetted up and down to kix.  
9. The two tubes were spun for a second time at 12.5xg for 3 minutes.  
10. The supernatant was removed using a bulb pipette and 1 mL of isopropanol was added and mixed by pipetting up and down or flicking   
11. The tubes were spun for a third time at 12.5 xg for 3 minutes  
12. The supernatant was removed, and 1 mL of isopropanol, and mixed by pipetting up and down.  
13. Pipetted the solution into a column syringes under a vacuum hood, and removed the supernatant in this process.  
14. The column syringes were removed and placed into two new microcentrifuge tubes and the syringes were removed leaving the columns attached to the top of tube. 
15.  The two tubes were centrifuged at 12 xg for 5 minutes.  
16.  The columns were transferred to a new microcentrifuge tube and 100 uL of Elution Buffer was added.  
17. The solution was let to sit for a minute, then spun at 12xg for one minute.  
18. The column was removed, and the solution was added to a microcentrifuge tube and stored in the freezer.  
19. Two plates were created for the spot titer test using 4 mL of LB Broth, 45 uL of CaCl2, and 5 mL of Top Agar.  
20. 4.5 mL of solution was added to each of the two 0.5 mL of Arthrobacter and poured onto the plates to let to set.  
21. Serial dilutions for a spot test were created using lysate from webbed plates. 
22. Eight microcentrifuge tubes were obtained and labeled 10^-1 to 10^-8.  
23. 90 uL of Phage buffer was added to each of the eight tubes.  
24. 10 uL of lysate was added to the 10^-1 serial dilution. The tube was vortexed and 10 uL of the 10^-1 serial dilution was added to the 10^-2 serial dilution tube.  
25. Continued this process until a serial dilution from 10^-1 to 10^-8 was obtained.  
26. 5 uL from each dilution was pipetted onto the respective portion of the plate.  
27. The plates were left to set for 10 minutes.  
28. The plates were place upright into the incubator.  

Observations/ Results: 

• Some of the plates for the spot titer test contained bubbles.  
• While performing DNA Extraction the pellet appeared cloudy.  

Next Steps/ Conclusions: 

Next class we will use the solution containing DNA to perform PCR. Using the results from the spot titer, the titer of the lysate will be determined.