Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.14 Mutagen Concentration Test - Seventh Protocol
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* Place all the tubes on the shaker at 37 Celsius. | * Place all the tubes on the shaker at 37 Celsius. | ||
- | * After 20 minutes, take only the tube labeled 0 from the shaker. Take 1mL from this tube and pipette it into a cuvette labeled 0. Using 1mL of LB in a cuvette, blank the spectrophotometer at 600 OD. Then measure the absorbance of the cuvette labeled 0. ''(In the actual procedure, 2 readings were done on the tube labeled 0. This means that the tube labeled 0 only had 8mL in it when the phage was added, which will slightly alter the phage to bacteria ratio for that tube.)'' | + | * After 20 minutes, take only the tube labeled 0 from the shaker. Take 1mL from this tube and pipette it into a cuvette labeled 0. Using 1mL of LB in a cuvette, blank the spectrophotometer at 600 OD. Then measure the absorbance of the cuvette labeled 0. ''(In the actual procedure, 2 readings were done on the tube labeled 0 to check for consistency. This means that the tube labeled 0 only had 8mL in it when the phage was added, which will slightly alter the phage to bacteria ratio for that tube.)'' |
* Remove the other two tubes after 30 minutes of incubation and add 87ul of T7 phage from the "10ul 7.19 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 two tubes after 30 minutes of incubation and add 87ul of T7 phage from the "10ul 7.19 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.)'' |
Revision as of 18:36, 14 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 (7.29)
3) Spot test to determine phage concentration
2) Applying the mutagen
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