Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/CsClGradientPhagePurification

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Revision as of 20:24, 5 June 2013

Phage Purification March - April Notebook: Experiments

5.15 Titer Test on 5.3 T7 new Phage Stock

I) Purpose

Determine the concentration of the 5.3 T7 new Phage Stock to give an estimate for starting mutagenesis

II) Expected Outcome

As the dilution series increases, the number of plaques on the plates should decrease

III) Reagants Used

E5.3 T7 new phage stock; LB; BL21 E. coli from 5.14 overnight

IV) Actual Procedure

Performed dilution series with 5.3 T7 stock; went down to -8

Add 0.5 mL of BL21 to 4 test tubes, labelled 5, 6, 7, 8

Add 20 uL of T7 new to each test tube. Phage concentration should correspond to the number on the test tube. Incubate at room temp for 20 minutes.

In a 50mL centrifuge tube, combine 10mL of LB with 10mL of x2 top agar.

Add 5mL of the combined solution to each of the four test tubes. Plate each tube and incubate at 37 C for 24 hours

V) Results

Lots of contamination - from the plates -> this batch of LB plates have been completely disposed of.

-8 plate had 7 plaques: This gives a phage concentration estimate of approximately 7E8 particle/20uL

-5, -6, and -7 had overlapping plaques

@@ Line 30: / Line 30: @@
 <font face="Calibri" size="3">
-<font size="5"> '''6.3 CsCl Gradient Phage Purification''' </font>
+<font size="5"> '''5.15 Titer Test on 5.3 T7 new Phage Stock''' </font>
 <br>
-. Primary ideas
-: Direct evolution method
+'''I) Purpose'''
+: Determine the concentration of the 5.3 T7 new Phage Stock to give an estimate for starting mutagenesis
-:: Separation method: ultracentrifugation sedimentation with sucrose gradient, size exclusion chromatography, TEM
+'''II) Expected Outcome'''
+: As the dilution series increases, the number of plaques on the plates should decrease
-:: Environment that would hasten evolution?
+'''III) Reagants Used'''
-::: Limit nutrient as Dr. Kooyman suggested – affect host more than phage?
+: E5.3 T7 new phage stock; LB; BL21 E. coli from 5.14 overnight
-::: Different strains of E Coli – will phage still infect?
-:: Selected phage can self replicate – storage
-::: Purified product and DNA sequence can direct further engineering for drug delivery purpose (protein team’s job)
-: Site-directed mutagenesis
+'''IV) Actual Procedure'''
-:: Identifying sites to mutate
-:: Modeling: compare CP and scaffolding protein sequence of similar phage that has slightly different size, deduce from this possible sites that can be mutated to produce smaller phage
-:: Capsid production
-::: Introduce genes into E coli for expression and assembly – producing a novel machine that is not naturally found
-:::: How many genes to introduce into E coli?
-:::: Stability?
-:: In vitro assembly?
-:::: Has been done with T7 genome in E coli extract (no cell)
-:::: Site directed mutagenesis of a genome (40kbp)
-:: Our parts would be plasmid introduced into E coli, but capsid produced this way won’t need extracting DNA/RNA from capsid to make an empty carrier
-<br>
+: Performed dilution series with 5.3 T7 stock; went down to -8
+: Add 0.5 mL of BL21 to 4 test tubes, labelled 5, 6, 7, 8
-. Refined plans
+: Add 20 uL of T7 new to each test tube. Phage concentration should correspond to the number on the test tube. Incubate at room temp for 20 minutes.
+: In a 50mL centrifuge tube, combine 10mL of LB with 10mL of x2 top agar.
-: Get the E coli and T7/Q beta to grow
+: Add 5mL of the combined solution to each of the four test tubes. Plate each tube and incubate at 37 C for 24 hours
-: Test with different Agar concentration – see if selection method works (evolution)
-:: Sequencing if evolution occurs
-: Compare genome to find targets for point mutation
-: Playing around with phage genome
-:: Knocking out the polymerase
-:: Change genome size - experiment – could go bigger or smaller
-: T7 specific
-:: Play around with minor and major capsid protein – CCC site
+'''V) Results'''
+: Lots of contamination - from the plates -> this batch of LB plates have been completely disposed of.
+: -8 plate had 7 plaques: This gives a phage concentration estimate of approximately 7E8 particle/20uL
+: -5, -6, and -7 had overlapping plaques
 </font>