November
30
NOVEMBER 26TH AND 28TH- Labs
- NOVEMBER 26TH, 2018
- OBJECTIVE:
- Plate high titer in order to flood plates and get more lysate for DNA extraction
- PROCEDURE:
- Tables were cleaned and lamps were lit
- The dilutions from last lab were used, and the following amounts were placed into a tube with .5mL of Arthrobacter for 15 minutes:
- 10^-2 45𝝁L
- 10^-2 45𝝁L
- 10^-1 4.5𝝁L
- During the 15 minutes a large test tube was filled with:
- 8mL LB
- 90𝝁L CaCl2
- 10mL 2X TA
- Then 4.5mL of the 2X TA solution was pipetted into the tube containing lysate and Arthrobacter, then the tube was poured onto a plate
- The previous step was repeated for all the tubes containing lysate and Arthrobacter
- For the control 4.5mL of the 2X TA solution was pipetted onto a plate
- The plates were then left to solidify for 10 minutes, before being inverted and placed into the incubator
- RESULTS:
- The Results can be viewed in Figure 26
- CONCLUSION:
- Minimal to no plaques grew due to how old the lysate was. As the lysate ages, the bacteriophage most likely lost infectivity.
- NEXT STEPS:
- Use original lysate to get a high titer plate
- OBJECTIVE:
- NOVEMBER 28TH, 2018
- OBJECTIVE:
- Use lysate from flooded plate (on 12/19) to create serial dilutions and get a high titer
- PROCEDURE:
- Tables were cleaned and lamps were lit
- Six micro-centrifuge tubes were labeled according to the dilution (10^0 through 10^-5)
- The tube labeled 10^0 was filled with 100𝝁L of phage buffer and tubes 10^-1 through 10^-6 were filled with 90𝝁L of phage buffer
- 100𝝁L of lysate was pipetted into the 10^-0 micro centrifuge tube
- Then 10𝝁L of the 10^0 dilution was pipetted into the 10^-1 tube, then 10𝝁L of solution from the 10^-1 tube was pipetted into the 10^-2 tube, and so on until a dilution of 10^-5 was reached
- The following amounts of dilutions were placed into a tube with .5mL of Arthrobacter for 15 minutes:
- 10^-2 45𝝁L
- 10^-4 10𝝁L
- 10^-1 4.5𝝁L
- 10^-2 10𝝁L
- 10^-5 10𝝁L
- During the 15 minutes a large test tube was filled with:
- 14mL LB
- 157.5𝝁L CaCl2
- 17.5mL 2X TA
- Then 4.5mL of the 2X TA solution was pipetted into the tube containing lysate and Arthrobacter, then the tube was poured onto a plate
- The previous step was repeated for all the tubes containing lysate and Arthrobacter
- For the control 4.5mL of the 2X TA solution was pipetted onto a plate
- The plates were then left to solidify for 10 minutes, before being inverted and placed into the incubator
- RESULTS:
- Waiting for results
- CONCLUSION:
- Waiting for results
- NEXT STEPS:
- Wait for results, if high titer was not achieved a TEM will be ran on lysate available, if high titer is achieved plates will be flooded and DNA extraction will be run
- Wait for results, if high titer was not achieved a TEM will be ran on lysate available, if high titer is achieved plates will be flooded and DNA extraction will be run
- OBJECTIVE:
