Team:BYU Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU Provo/5.3 T7 phage selection method test

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Revision as of 15:32, 24 May 2013

5.3 T7 Phage Selection Method Test

I) Purpose

Test the size of plaques formed from x6 and x8 top agar

II) Expected outcome

As top agar increase in concentration, the number of plaques/plate should decrease and individual plaques should become smaller.

III) Reagent record

T7 + phage: -5 dilution from 4.26; E coli BL21; Agar: ×2 prepared by Jordan in 500mL glass bottle; LB: prepared by us on 4.3; overnight bacteria culture: set up on Thursday at 2:00pm using 4.6 streak; ×8 top agar stock: prepared by us on 4.3

IV) Actual procedures / observations

i) Six test tubes were labeled control, ×6, ×6, ×8, ×8, x8, and ×8. To each of the test tubes, 0.5mL of E coli overnight liquid culture was added.

ii) To each test tube 20 μL of -5 phage solution was added. This mixture of E coli and phage was left sitting for 20 minutes before proceeding.

iii) In a separate set of test tubes, top agar was diluted to give ×6, and ×8 concentration.

iv) 5mL of top agar was added to each test tube so that the concentration of the agar added matches the labels on test tube. For the control tube, ×8 agar was added. The contents of these test tubes were then plated on LB plates. Note: ×6 and ×8 solidified as soon as the content hit the plate. As a result, top agar wasn’t spread evenly.

v) After the top agar has solidified, the plates were incubated up side down

V) Results

The x8 plates had smaller and few plaques than the x6 plates

The x8 plaques are about 1mm in diameter

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 {{TeamBYUProvo}}
-===5.3 Phage Amplification/Purification===
+===5.3 T7 Phage Selection Method Test===
 '''I) Purpose'''
-: To create a higher titer of phage solution
+: Test the size of plaques formed from x6 and x8 top agar
+'''II) Expected outcome'''
+: As top agar increase in concentration, the number of plaques/plate should decrease and individual plaques should become smaller.
+'''III) Reagent record'''
+: T7 + phage: -5 dilution from 4.26; E coli BL21; Agar: ×2 prepared by Jordan in 500mL glass bottle; LB: prepared by us on 4.3; overnight bacteria culture: set up on Thursday at 2:00pm using 4.6 streak; ×8 top agar stock: prepared by us on 4.3
+'''IV) Actual procedures / observations'''
+: i) Six test tubes were labeled control, ×6, ×6, ×8, ×8, x8, and ×8. To each of the test tubes, 0.5mL of E coli overnight liquid culture was added.
+: ii) To each test tube 20 μL of -5 phage solution was added. This mixture of E coli and phage was left sitting for 20 minutes before proceeding.
+: iii) In a separate set of test tubes, top agar was diluted to give ×6, and ×8 concentration.
+: iv) 5mL of top agar was added to each test tube so that the concentration of the agar added matches the labels on test tube. For the control tube, ×8 agar was added. The contents of these test tubes were then plated on LB plates. ''Note: ×6 and ×8 solidified as soon as the content hit the plate. As a result, top agar wasn’t spread evenly.''
+: v) After the top agar has solidified, the plates were incubated up side down
-'''II) Expected Outcome'''
+'''V) Results'''
-: A purified high titer of phage
+: The x8 plates had smaller and few plaques than the x6 plates
+: The x8 plaques are about 1mm in diameter
-'''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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