5.3 T7 phage amplification/purification

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(Created page with "===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) Re...")
 
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===5.3 Phage Amplification/Purification===
===5.3 Phage Amplification/Purification===
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'''IV) Actual Procedure/Observations'''
'''IV) Actual Procedure/Observations'''
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Set up phage liquid culture (4.27)
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 + 100μL phage stock (0, original received)
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: 4) Add 100 uL of chloroform to each of the new tubes and gently shake
: 4) Add 100 uL of chloroform to each of the new tubes and gently shake
: 5) We labeled the purified stock 5.3
: 5) We labeled the purified stock 5.3
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Spot test (5.6)
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Spot test (5.5)
: 1) Create a 1:10 dilution series from 0 to -11 of liquid culture that was purified above
: 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
: 2) Put 1mL of E.Coli in a 50mL centrifuge tube
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: 5) Spot 5uL of each dilution onto the two plates, 6 on each. The labels on the plates should correspond to dilution series number
: 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  
: 6) Incubate overnight at 37 C  
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V) Results
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'''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.
: From spot test, we know that our phage amplification procedure worked somewhat. We were able to increase phage titer to approximately -7 or -8.
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VI) Proposed next step
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'''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.
: 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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Latest revision as of 21:41, 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 (5.5)

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.

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