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7.13 Phage Propagation Experiment
I) Purpose
- To amplify the 5.3 phage stock solution we have been using for mutagenesis and determine an accurate titer for future mutagenesis.
II) Expected Outcome
- A purified high titer of phage. The new stock should have a higher titer than the 5.3 phage stock, which decreased in phage viability due to sitting in the fridge for 2 month.
III) Reagent Record
- Chloroform from Dr. Grose’s lab; LB; E coli BL21 liquid culture overnight; x8 top agar; 5.3 phage stock
IV) Actual Procedure/Observations
- 1. Set up phage liquid culture (7.13)
- - phage: 4mL LB + 1mL E coli
- 10uL phage: 4mL LB + 1mL E coli + 10μL 5.3 phage stock
- 100uL phage: 4mL LB + 1mL E coli + 100μL 5.3 phage stock
- :1mL phage: 4mL LB + 1mL E coli + 1mL 5.3 phage stock
- 2. Liquid culture purification (7.15)
- 1) For each infected test tubes (10uL, 100uL, 1mL), put 1mL of liquid culture into 5 eppendorf tubes.
- 2) Centrifuge at 3000 rpm for 5 minutes
- 3) Transfer supernatant into new eppendorf tubes. 5 eppendorf tubes for each phage concentration
- 4) Add 100 uL of chloroform to each of the new tubes and gently shake
- 5) We labeled the purified stock as 7.15
- 3. Titer to determine phage concentration (7.15)
- 1) For each concentration of phage, specifically 10uL, 100uL, and 1mL, 1:100 dilution series were performed to generate 0, -2, -4, -6. 1:10 dilution series were then performed to generate -7, -8, and -9.
- 2) 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.
- 3) Plates were incubated upside down for 24 hours.
V) Results
- 1. Set up phage liquid culture
- 2. Liquid culture purification
- 1) For each infected test tubes (10uL, 100uL, 1mL), put 1mL of liquid culture into 5 eppendorf tubes.
- 2) Centrifuge at 3000 rpm for 5 minutes
- 3) Transfer supernatant into new eppendorf tubes. 5 eppendorf tubes for each phage concentration
- 4) Add 100 uL of chloroform to each of the new tubes and gently shake
- 5) We labeled the purified stock as 7.15
- 3. Titer to determine phage concentration
- 1) For each concentration of phage, specifically 10uL, 100uL, and 1mL, 1:100 dilution series were performed to generate 0, -2, -4, -6. 1:10 dilution series were then performed to generate -7, -8, and -9.
- 2) 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.
- 3) Plates were incubated upside down for 24 hours.
VI) Proposed next step
- We’ll use this stock to further concentrate phage. Testing whether the amount of phage added to liquid culture will affect phage titering result will be helpful.
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