Team:BYU Provo/Notebook/SmallPhage/Summerexp/9.13 Mutagen Concentration Test
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* Label 4 test tubes C, 0, 500, 1000. 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. | * Label 4 test tubes C, 0, 500, 1000. 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 500ul of mutagen to tube labeled 500) | + | * 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 500ul of mutagen to tube labeled 500). Don't add mutagen to C. |
* Place all the tubes on the shaker at 37 Celsius. | * Place all the tubes on the shaker at 37 Celsius. | ||
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* After 20 minutes, take only the tube labeled C from the shaker. Take 1mL from this tube and pipette it into a cuvette labeled C. Using 1mL of LB in a cuvette, blank the spectrophotometer at 600 OD. Then measure the absorbance of the cuvette labeled C. ''(In the actual procedure, 2 readings were done on the tube labeled C to check for consistency.'' | * After 20 minutes, take only the tube labeled C from the shaker. Take 1mL from this tube and pipette it into a cuvette labeled C. Using 1mL of LB in a cuvette, blank the spectrophotometer at 600 OD. Then measure the absorbance of the cuvette labeled C. ''(In the actual procedure, 2 readings were done on the tube labeled C to check for consistency.'' | ||
- | * Remove the other three tubes after 30 minutes of incubation and add AMOUNT of T7 phage from the "8.24 stock" to each tube. ''(There should be a 1:10 phage to bacteria concentration. The calculations are as follows: The spectrophotometer 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. This means that we need '''2.175E8''' phage added to each tube. Because the 7.19 stock is concentrated at '''2.5E9 phage/ml [5E7 phage/20ul], 87ul''' of phage will provide '''2.175E8''' phage.)'' | + | * Remove the other three tubes after 30 minutes of incubation and add AMOUNT of T7 phage from the "8.24 stock" to each tube, except C. From this point on, tube C is irrelevant and can be discarded. ''(There should be a 1:10 phage to bacteria concentration. The calculations are as follows: The spectrophotometer 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. This means that we need '''2.175E8''' phage added to each tube. Because the 7.19 stock is concentrated at '''2.5E9 phage/ml [5E7 phage/20ul], 87ul''' of phage will provide '''2.175E8''' phage.)'' |
* Incubate all the tubes on the shaker at 37 Celsius for 80 minutes. | * Incubate all the tubes on the shaker at 37 Celsius for 80 minutes. |
Revision as of 21:34, 13 September 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) (9.13)
2) Applying the mutagen (9.14)
3) Spot test to determine phage concentration (8.16) Dilution series -2 through -7 were performed all three samples (0, 500, and 1000). These dilutions samples were then used in spot tests to estimate phage titer.
V) Results 2) Applying the mutagen
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