Team:BYU Provo/Notebook/SmallPhage/Springexp/Period3/Exp/7.29 Mutagen Concentration Test - Sixth Protocol

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Small Phage July - August Notebook: Experiments

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

@@ Line 54: / Line 54: @@
 : - Uracil solution (2.5mg/mL)
-: - Adenine solution (5mg/mL
+: - Adenine solution (5mg/mL)
 : - '''7.15 phage stock'''
@@ Line 60: / Line 60: @@
 : - '''x8''' top agar
-: - 102mL of LB
+: - '''102mL''' of LB
 '''IV) Procedure'''
@@ Line 66: / Line 66: @@
 ) Overnight (The day before) (7.28)
-: - Add '''12mL''' of LB into a test tube and add 1 colony of E. Coli BL21 into the tube using a wooden stick.
+: - Add 5mL of LB into a test tube and add 1 colony of E. Coli BL21 into the tube using a wooden stick.
 ) 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 '''9mL''' of LB, '''40ul''' of adenine solution, and '''80ul''' of uracil solution into each test tube. Then add '''1mL''' of BL21 overnight into each test tube. Incubate on the shaker at 37 Celsius.
+: - 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+.
-: - Remove all the test tubes off the shaker after '''2 hours'''. Centrifuge the M9 labeled tubes at 4000rpm for 10 minutes at 7 Celsius. Discard the supernatant using pipettes. Add 10mL of the M9 solution into each M9 labeled tube with a pipette, being sure to pipette up and down to resuspend the bacteria.
+: - 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 90 minutes.
+: - Place all the tubes on the shaker at 37 Celsius for 30 minutes.
-: - Remove the tubes and add '''AMOUNT''' of T7 phage from the '''7.15''' stock to each tube, except for the tubes with a "C" on them.
+: - 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 '''1 hour'''.
+: - 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.