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7.29 Mutagen Concentration Test - Sixth Protocol
I) Purpose
- - To test the sixth protocol for applying 5-bromodeoxyuridine and inducing mutation. We also hope to see if using LB or M9 is better for the mutagen process.
II) Expected Outcome
- - A decrease in phage viability with increasing mutagen concentration.
- - A few random mutations that produce smaller and larger phage that can be isolated.
- - M9 media should allow E. Coli BL21 to take in more of the mutagen so there should be more mutations in T7 phage, resulting in a smaller titer when compared with LB media.
III) Reagents Used
- - 5-bromodeoxyuridine stock in freezer labeled T7 (10mg/mL)
- - 50mL of M9+ prepared by the large phage group
- - Uracil solution (2.5mg/mL)
- - Adenine solution (5mg/mL)
- - 7.15 phage stock
- - x8 top agar
- - 102mL of LB
IV) Procedure
1) Overnight (The day before) (7.28)
- - Add 5mL of LB into a test tube and add 1 colony of E. Coli BL21 into the tube using a wooden stick.
2) Applying the mutagen (7.29)
- - Label 10 test tubes LB:C, LB:0, LB:100, LB:200, LB: 500, M9:C, M9:0, M9:100, M9: 200, and M9: 500. Add 9.9mL of LB, 40ul of adenine solution into each test tube. Then add 100ul of BL21 overnight into each test tube. Incubate on the shaker at 37 Celsius.
- - Make the M9+ suspension medium and supplement it with the appropriate amount of uracil and adenine. In this case, 200uL of adenine solution and 400uL of uracil solution was add 50mL of M9+.
- - Make a LB/adenine/uracil solution by adding 200uL of adenine solution and 400uL of uracil solution to 50mL of LB.
- - Remove all the test tubes off the shaker after 2 hours. Centrifuge all the tubes at 4000rpm for 10 minutes at 7 Celsius. Discard the supernatant using pipettes. For the LB tubes, add 10mL of the LB/adenine/uracil solution into each tube with a pipette, being sure to pipette up and down to resuspend the bacteria. For the M9 tubes, add 10mL of the M9 solution into each M9 labeled tube with a pipette and resuspend the bacteria.
- - To each tube, add the corresponding amount in ul of 5-bromodeoxyuridine, a mutagen, to each test tube with a 100, 200, or 500. (Ex: Add 100ul of mutagen to LB:100 and M9:100.)
- - Place all the tubes on the shaker at 37 Celsius for 30 minutes.
- - Remove the tubes and add AMOUNT of T7 phage from the "10ul 7.19 stock" to each tube, except for the tubes with a "C" on them.
- - Incubate all the tubes on the shaker at 37 Celsius for 80 minutes.
- - Remove all the tubes (place tubes with a "C" to the side; they are no longer needed) and add 1mL of chloroform to each. Gently shake each tube and centrifuge it at 4000rpm for 10 minutes at 7 Celsius. Remove the supernatant from each tube with a pipette and place it in a new tube with the same label. Be careful not to get the chloroform or bacteria when you remove the supernatant.
- - Store the supernatants at 4 Celsius.
V) Results
VI) Conclusion
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