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5.3 T7 phage amplification/purification
From 2013.igem.org
5.3 Phage Amplification/Purification
I) Purpose
- To create a higher titer of phage solution
II) Expected Outcome
- A purified high titer of phage
III) Reagent Record
- Chloroform from Dr. Grose’s lab. Liquid cultures prepared on 4.27
IV) Actual Procedure/Observations
Set up phage liquid culture (4.27)
- + phage: 4mL LB + 1mL E coli + 100μL phage stock (0, original received)
- - phage: 4mL LB + 1mL E coli
Liquid culture purification
- 1) Put 1mL of liquid culture into 4 eppendorf tubes
- 2) Centrifuge at 3000 rpm for 5 minutes
- 3) Transfer supernatant into 4 new eppendorf tubes
- 4) Add 100 uL of chloroform to each of the new tubes and gently shake
- 5) We labeled the purified stock 5.3
Spot test (5.6)
- 1) Create a 1:10 dilution series from 0 to -11 of liquid culture that was purified above
- 2) Put 1mL of E.Coli in a 50mL centrifuge tube
- 3) Add 5mL LB with 5mL of x2 top agar to the 50mL centrifuge tobe
- 4) Plate 5mL of top agar solution in centrifuge tube onto 2 plates
- 5) Spot 5uL of each dilution onto the two plates, 6 on each. The labels on the plates should correspond to dilution series number
- 6) Incubate overnight at 37 C
V) Results
- From spot test, we know that our phage amplification procedure worked somewhat. We were able to increase phage titer to approximately -7 or -8.
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.
