Rationale:

TEM and DNA Extraction were performed since a known medium titer was used to create a new lysate and enough lysate was obtained to start on DNA Extraction.  

Procedure:  

1. An aseptic zone was created using 70% ethanol, Cidecon, and an ethanol burner.  
2. For DNA extraction, 10 mL of lysate were obtained and pipetted into a 50 mL tube.  
3. 40 uL of Nuclease was added into the 50 mL tube and flipped 10 times.  
4. 4 mL of PEG was added into the 50 mL tube inverted once   
5. The 50 mL tube was placed in the incubator for 25 minutes.  
6. While waiting the 25 minutes, TEM grid was made by adding 20 uL of lysate, two 20 uL of DI water, and about 20 uL of Uranyl acetate on parafilm.  
7. A small mesh copper grid was obtained and placed shiny side down in the lysate for five minutes.  
8. The 50 mL tube for DNA extraction was moved to sit at room temperature for 40 minutes.  
9. After the five minutes in the lysate using forceps removed copper TEM grid from lysate and set in DI water for 2.5 minutes.  
10. The TEM grid was moved to the next DI water for another 2.5 minutes.  
11. Then the TEM grid was moved to Uranyl Acetate for a minute.  
12. The TEM grid was then blotted dry and viewed under the electron microscope.  

Observation/Results: 

• Spot Titer Test lysed for serial dilutions from 10^-1 to 10^-3. The rest of the serial dilutions did not produce any phage, and therefore could not use this plate to determine the titer of the plate.
• 

  Spot Test Plate 1

  

  Spot Test Plate 2

• TEM and DNA Extraction were performed assuming that a high titer would be produced from a medium titer plate of 10^7 since 10^-1 to 10^-3 serial dilutions were lysed as well.  
  

  Phage on TEM

• Since the centrifuge machine had some technical difficulties, Dr. Adair centrifuged the lysate solution and separated the pellet from the supernatant and stored the pellet in the freezer. 

Next Step/ conclusions: 

Next class DNA Extraction will be completed to be used for PCR, and another spot titer test will be performed to calculate the titer of the plate.