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- Small Phage
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7.8 Mutagen Concentration Test - Fourth Protocol
I) Purpose
- To test the forth protocol for applying 5-bromodeoxyuridine and inducing mutation.
II) Expected Outcome
- - A decrease in phage viability with increasing mutagen concentration.
- - A few random mutations that produces smaller phage and consequently larger plaques during selection with x8 agar.
III) Reagents Used
- 5-bromodeoxyuridine stock in freezer labeled T7 (10mg/mL); 50mL of M9+ prepared by the large phage group; BL21 overnight; 5.3 phage stock; x8 top agar; uracil solution (2.5mg/mL); adenine solution (5mg/mL).
IV) Procedure
1) Applying the mutagen (7.8)
- - From 5.13 Determining E coli concentration with spectrophotometer, we know that the concentration of E coli BL21 in a liquid overnight culture is about 3.12E8 cell/mL. But we need to resuspend it in M9+ medium.
- - To make the M9+ suspension medium supplemented with the appropriate amount of uracil and adenine, 200uL of adenine solution and 400uL of uracil was add 50mL of M9+.
- - Label 5 centrifuge tubes (15mL) 0, 100, 200, 500, and C (control). To each of these test tubes, add 10 mL of E coli overnight. Cells were then pelleted via centrifuge at 4000rpm for 10min at 7 Celsius. Supernatant was taken out using pipets and discarded. E coli was then resuspended in 10mL M9+ supplemented with uracil and adenine.
- - To four centrifuge tubes, add 0, 100, 200, and 500 uL of 5-bromodeoxyuridine (add nothing to C). The volume of the mutagen should correspond to the label on the test tube. Because 5-bromodeoxyuridine stock is a 10mg/mL aqueous solution, the final mutagen concentration in the five test tubes will be 0, 100, 200, and 500 ug/mL.
- - Let the bacterial suspension incubate with mutagen for approximately 1h 30min.
- - 30uL of 5.3 phage stock was added to each centrifuge tube (add nothing to C). From 5.15 Titer Test on 5.3 T7 new Phage Stock, we know that 5.3 phage stock has 7E8 particles/20uL. Thus, to each of the test tubes, we are adding approximately 1.2E9 particles.
- - Incubate phage on shaker at 37C for 3 hours.
- - Added 1mL of chloroform to each test tube. Liquid culture from the four test (excluding C) tubes is then centrifuged at 4000 rpm for 10 minutes to pellet cell debris. Supernatant containing phage particles is removed and placed in a new tube.
- - The stock solutions are now stored in 4 Celsius.
2) Titer to Determine Phage Concentration 1 (7.10)
- - For each mutagenesis, specifically 0, 100, 200, and 500ug/mL, 1:100 dilution series were performed to generate 0, -2, -4, -6, and -8 dilutions.
- - For each dilution, a test tube was assigned to it. To each test tube, 0.75mL of BL21 liquid culture overnight was added and then transfected with 30uL of phage dilution. After 20 minutes of incubation, 7mL of x8 agar was added to each test tube and the content was then plated.
- Plates were incubated upside down for 24 hours.
3) Spot Test to Confirm Phage Viability (7.12)
- - Add 0.75mL of BL21 overnight into 8 test tubes
- - Add 7mL of x6 top agar to each tube and plate it on top of LB plates
- - Divide the plates into 6 quadrants with a sharpie. 4 plates are for the new dilution (next step) and 4 plates are for the old dilution series (step 2), each labeled with their respective mutagen concentration series.
- - Using the mutagenized phage stock from step 1 above, create a 1:10 dilution series of each mutagen concentration (0ul, 100ul, 200ul, and 500ul) from 0 to -4 using 90ul LB and 10ul phage in eppendorf tubes.
- - Spot 5ul of each eppendorf tube (from both new and old dilution) onto the plates. Also spot 5ul of 5.3 phage stock on each plate as a control.
V) Results
2) Titer to Determine Phage Concentration 1
- - Only the 0 plates from the 100ul, 200ul, and 500ul series showed plaques. All other plates had no plaques. The 0 200ul plate had the most plaques.
3) Spot Test to Confirm Phage Viability
- - The old dilution series plates showed plaques at 0 and 5.3.
- - The new dilution series plates showed the following plaques:
- - 0ug: 5.3, 0
- - 100ug: 5.3, 0, -1
- - 200ug: 5.3, 0, -1, -2
- -500 ug: 5.3, 0
VI) Conclusion
- Our results don't point to any concrete conclusion. The results try to tell us that as we add mutagen up to 200ug, phage viability increases. At 500ug, phage viability drops. We know this can't be right, especially because our control (0ug) showed an abnormal decrease in phage. This points to a possible error in our experiment. The 3 possible errors we could find were 1) 5.3 stock might be dying, 2) The overnights had their lids screwed on tight so that might have killed off some of the bacteria, and 3) It's possible that when we started overnights in different test tubes, the concentrations of bacteria could have been different. We will correct these 3 practices in our next experiments.
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