Team:BYU Provo/Notebook/SmallPhage/Springexp/Period3/Exp/7.29 Mutagen Concentration Test - Sixth Protocol
From 2013.igem.org
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'''I) Purpose''' | '''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. | + | : - 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''' | '''II) Expected Outcome''' | ||
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: - 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 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. | + | : - 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. |
- | : 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.) | + | : - 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 90 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 '''7.15''' 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 '''1 hour'''. |
- | : 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. | + | : - 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. | + | : - Store the supernatants at 4 Celsius. |
Revision as of 22:21, 23 July 2013
Small Phage July - August Notebook: Experiments
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7.29 Mutagen Concentration Test - Sixth Protocol
I) Purpose
II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Overnight (The day before) (7.28)
2) Applying the mutagen (7.29)
VI) Conclusion
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