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Revision as of 18:25, 21 May 2013

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.