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Revision as of 22:26, 24 June 2013
| Phage Purification March - April Notebook: Experiments
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- Overview
- March-April
- May-June
- July-August
- September-October
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4.5 Phage Spot Test
I) Purpose
- Choose a bacterium to grow phage in.
II) Expected Outcome
- Larger plaques in the bacteria where the phage were able to travel and infect the best.
III) Reagants Used
- BL21
- W3110
- LB
- 1 X LB Top Agar (TA)
- Phage:
- 1L
- 10L
- 40T4
- T4DOS
- T4 Infected
IV) Actual Procedure
- We placed 100 µL of LB broth into 6 tubes, labeled 0 to -5. We repeated this for all 5 strains of phage. Next, 10 µL phage was transferred to the tube labeled '0'. The tube was vortexed and 10µL was transferred to the next tube. this was repeated to the concentration of -5.
- While performing this procedure, we had innoculated .5 mL LB broth with W3110, and another .5 mL culture with BL21. This was done six times for each bacterium, so each phage type would have it's own plate split into 6 concentrations of phage.
- Next we plated the bacteria using 4.5 mL TA and .5 Bacterial cultures. The plates were segmented, and we spotted 5µL of the different concentrations of phage onto the various segments of the plates.
- To finish, the plates were incubated overnight at 37°C.
V) Results
- Phage 10L w/ w3110 had large scale infection every concentration
- Phage 10L w/ BL21 had infection in very large concentrations
- Phage 1L w/ w3110 had infection in very large concentrations
- Phage 1L w/ BL21 had infection in very large concentrations
- Phage T4 DOS w/ w3110 had infection in very large concentrations
- Phage T4 DOS w/ BL21 had infection in very large concentrations
- Phage 40 T4 w/ w311o had no phage infection
- Phage 40 T4 w/ BL21 had large scale infection at every concentration
- Phage T4 inf w/ w311o had large scale infection at every concentration
- Phage T4 inf w/ BL21 had large scale infection at every concentration
- We had a significant amount of running that occurred on almost every plate, so the results were a little difficult to read. We concluded that the phage is a high enough concentration that we would have to do another phage titer to a smaller dilution in order to determine actual concentration. The controls were all a lawn of bacteria.
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