Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.14 Mutagen Concentration Test - Seventh Protocol
From 2013.igem.org
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1) Overnight (The day before) (8.13) | 1) Overnight (The day before) (8.13) | ||
- | * Add 5mL of LB into a test tube and add 1 colony of E. Coli | + | * Add 5mL of LB into a test tube and add 1 colony of E. Coli B into the tube using a wooden stick. |
- | 2) Applying the mutagen ( | + | 2) Applying the mutagen (8.14) |
- | * Label 3 test tubes 0, 200, 500. Add 9.8mL of LB and 40ul of adenine solution into each test tube. Then add 200ul of | + | * Label 3 test tubes 0, 200, 500. Add 9.8mL of LB and 40ul of adenine solution into each test tube. Then add 200ul of E coli B overnight into each test tube. Incubate on the shaker at 37 Celsius. |
* Remove all the test tubes off the shaker after 2 hours. Add 40ul of adenine and 80ul of uracil to each test tube. Also add the corresponding amount in ul of 5-bromodeoxyuridine, a mutagen, to each test tube. (Ex: Add 200ul of mutagen to tube labeled 200) | * Remove all the test tubes off the shaker after 2 hours. Add 40ul of adenine and 80ul of uracil to each test tube. Also add the corresponding amount in ul of 5-bromodeoxyuridine, a mutagen, to each test tube. (Ex: Add 200ul of mutagen to tube labeled 200) | ||
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* Store the supernatants at 4 Celsius. | * Store the supernatants at 4 Celsius. | ||
- | 3) Spot test to determine phage concentration | + | 3) Spot test to determine phage concentration (8.14) |
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+ | * Dilution series -2 through -7 were performed all three samples (0, 200, and 500). These dilutions samples were then used in spot tests to estimate phage titer. | ||
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+ | * Specifically, three plates were prepared for spot tests using E. coli B by adding 0.5mL overnight into a tube, mixing it with 5mL of x1 top agar, and then plating the content. 5ul of each dilution sample was spotted onto corresponding plate. | ||
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+ | 4) CsCl gradient (8.15) | ||
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+ | * Phage Purification Team was able to finalize their procedures for performing CsCl gradient on T7 phage. For specifics please refer to '''add link''' | ||
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+ | * The finished gradient was aliquoted into 27 eppendorf tubes, each containing 0.5-1mL solution. We will need to determine phage characteristics in each aliquot before proceeding with dialysis and TEM. | ||
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+ | 5) Characterizing post-CsCl phage (8.16-8.18) | ||
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+ | * Spot test was performed to determine which aliquots contain viable phage. Specifically, after plating 0.5mL of E coli B overnight and 5mL of x1 agar, 10uL of each aliquot was spotted onto the plates. Plaques formation would indication a phage concentration of at least 10E2 pfu/mL. | ||
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* The OD reading was 0.435A, which indicates there are 2.175E8 bacteria/ml. Because there are 10ml in each tube, there is roughly 2.175E9 bacteria per tube. | * The OD reading was 0.435A, which indicates there are 2.175E8 bacteria/ml. Because there are 10ml in each tube, there is roughly 2.175E9 bacteria per tube. | ||
+ | 3) Spot test to determine phage concentration | ||
+ | * 0 went down to -5; 200 went down to -5; 500 went down to -6. | ||
Revision as of 21:23, 17 August 2013
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8.14 Mutagen Concentration Test - Seventh Protocol
I) Purpose To mutate T7 phage for different capsid sizes. II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Overnight (The day before) (8.13)
2) Applying the mutagen (8.14)
3) Spot test to determine phage concentration (8.14)
4) CsCl gradient (8.15)
5) Characterizing post-CsCl phage (8.16-8.18)
2) Applying the mutagen
3) Spot test to determine phage concentration
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