Team:BYU Provo/Notebook/SmallPhage/Springexp/Period3/Exp/6.27 Mutagen Concentration Test - Third Protocol
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: - 30uL of 5.3 phage stock was added to each centrifuge tube (add nothing to C). ''From 5.15 Titer Test on 5.3 T7 new Phage Stock, we know that 5.3 phage stock has 7E8 particles/20uL. Thus, to each of the test tubes, we are adding approximately 1.2E9 particles.'' | : - 30uL of 5.3 phage stock was added to each centrifuge tube (add nothing to C). ''From 5.15 Titer Test on 5.3 T7 new Phage Stock, we know that 5.3 phage stock has 7E8 particles/20uL. Thus, to each of the test tubes, we are adding approximately 1.2E9 particles.'' | ||
- | : - Incubate phage on shaker at 37C until clearage | + | : - Incubate phage on shaker at 37C until clearage: 22hours. |
: - Liquid culture from the four test (excluding C) tubes is then centrifuged at 4000 rpm for 10 minutes to pellet cell debris. Supernatant containing phage particles is removed and 1mL of chloroform is added to destroy any remaining E coli cells. | : - Liquid culture from the four test (excluding C) tubes is then centrifuged at 4000 rpm for 10 minutes to pellet cell debris. Supernatant containing phage particles is removed and 1mL of chloroform is added to destroy any remaining E coli cells. | ||
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: - The stock solutions are now stored in 4 Celsius. | : - The stock solutions are now stored in 4 Celsius. | ||
+ | 2) Titer to determine phage concentration (6.28) | ||
+ | |||
+ | : - For each mutagenesis, specifically 0, 100, 200, and 500ug/mL, 1:100 dilution series were performed to generate 0, -2, -4, -6, and -8 dilutions. | ||
+ | |||
+ | : - For each dilution, a test tube was assigned to it. To each test tube, 0.75mL of BL21 liquid culture overnight was added and then transfected with 30uL of phage dilution. After 20 minutes of incubation, 7mL of x8 agar was added to each test tube and the content was then plated | ||
'''V) Results''' | '''V) Results''' | ||
+ | 1) Applying the mutagen | ||
+ | |||
+ | : - No clearage was seen after 22 hours. Following steps were carried regardless. | ||
+ | |||
+ | 2) Titer to determine phage concentration | ||
Revision as of 18:27, 28 June 2013
Small Phage May - June Notebook: Experiments
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6.27 Mutagen Concentration Test - Third Protocol
I) Purpose
II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Applying the mutagen (6.12)
2) Titer to determine phage concentration (6.28)
V) Results 1) Applying the mutagen
2) Titer to determine phage concentration
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