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7.17 Mutagen Concentration Test - Fifth Protocol
I) Purpose
- To test the fifth 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; 5.15 phage stock; x8 top agar; uracil solution (2.5mg/mL); adenine solution (5mg/mL).
IV) Procedure
1) Applying the mutagen (7.17)
- - 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+.
- - Combine the BL21 overnights so that there is 70mL in one tube. This allows for an equal bacterial concentration in each tube in the next step.
- - Label 7 centrifuge tubes (15mL) 1, 1C, 3, 3C, 16, 16C, and C ("C" is for 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 3 centrifuge tubes (1, 3, and 16), add 200 uL of 5-bromodeoxyuridine (add nothing to C). 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.
- - 90ul of 7.17 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 6.5E9 particles.
- - Incubate phage on shaker at 37C for for 1 hour, 3 hours, and 16 hours, respectively.
- - At each time point, pull the 2 corresponding tubes off the shaker (Ex: At 1 hour, take the 1 and 1C tubes.) Add 1mL of chloroform to each test tube. Liquid culture from the test 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) Spot Test to Confirm Phage Viability (7.19)
- - Add 0.75mL of BL21 overnight into 6 test tubes
- - Add 7mL of x1 top agar to each tube and plate it on top of the 6 LB plates
- - Divide the plates into 6 quadrants with a sharpie. Label the quadrants 7.15, -4, -5, -6, -7, and -8.
- - Using the mutagenized phage stock from step 1 above, create a 1:100 dilution series of the 6 solutions in step 1 for -2 and -4 in eppendorf tubes. Then make a 1:10 series for -5, -6, -7, and -8.
- - Spot 5ul of each eppendorf tube (-4 through -8) onto the plates. Also spot 5ul of 7.15 phage stock on each plate as a control.
V) Results
2) Spot Test to Confirm Phage Viability
- From the spot tests, 1 and 3C had a plaque at -4 (The plaque on -4 for 16 was incorrect, as we accidently added -2 to that spot). All plates have plaques for the 7.15 control.
VI) Conclusion
It appears as though incubating the phage with mutagen for 1 hour is better than 3 hours and 16 hours; however, we cannot hold this to be terribly accurate because the control for 1 hour showed no plaques at -4, suggesting that there might be something wrong with the experiment.
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