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

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Latest revision as of 00:27, 28 September 2013

Phage Purification May - June Notebook: Experiments

Phage Purification

6.3 CsCl Gradient Phage Purification

I) Purpose

Further purify the phage to a high level of purification.

II) Expected Outcome

Purified and viable phage will be extracted from the CsCl gradient.

III) Reagants Used

T4 and T7 purified phage from 5.26 PEG Purification

CsCl

phage suspension buffer

IV) Actual Procedure

Create different concentrations of CsCl solutions to create a gradient.

Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.

Add 4.92 g of CsCl to 6 ml of phage suspension buffer to create a 1.6 g/ml density gradient.

Layer two centrifuge tubes with 2 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 3 mL of 1.3 g/mL.

Layer T4 and T7 on top of the gradient in separate tubes.

Fill the remaining space in the tube with phage suspension buffer to 3-5 mL from the top.

Centrifuge at 26500 rpms (100,000 g) for 2.5 hours.

Leave overnight in the refrigerator.

V) Results

After centrifugation, we were able to see a distinct band of phage in both the T4 and T7 gradients. The T7 band was significantly lower in the tube than the T4 band. Next time we run the procedure, we will be running the T7 phage through a higher concentrated gradient. After leaving the gradients overnight in the refrigerator, the gradients mixed together and we had difficulty seeing the distinct bands to extract the phage. We will have to rerun the experiment.

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-| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification March - April Notebook: Experiments'''</font>
+| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification May - June Notebook: Experiments'''</font>
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-: [[Team:BYU_Provo/Small_Phage|Overview]]
+<font size = "4">
+: <u> '''Phage Purification''' </u> </font>
 : [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
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-| style="width: 82%; background-color: transparent;"|
+| style="width: 80%; background-color: transparent;"|
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 <br>
-. Primary ideas
-: Direct evolution method
+'''I) Purpose'''
+: Further purify the phage to a high level of purification.
-:: Separation method: ultracentrifugation sedimentation with sucrose gradient, size exclusion chromatography, TEM
+'''II) Expected Outcome'''
+: Purified and viable phage will be extracted from the CsCl gradient.
-:: Environment that would hasten evolution?
+'''III) Reagants Used'''
-::: Limit nutrient as Dr. Kooyman suggested – affect host more than phage?
+: T4 and T7 purified phage from [[Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period1/Exp/5.26 PEG Purification|5.26 PEG Purification]]
-::: Different strains of E Coli – will phage still infect?
+: CsCl
+: phage suspension buffer
-:: 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
-:: 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>
-. Refined plans
+'''IV) Actual Procedure'''
-: Get the E coli and T7/Q beta to grow
+: Create different concentrations of CsCl solutions to create a gradient.
-: Test with different Agar concentration – see if selection method works (evolution)
+:: Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
-:: Sequencing if evolution occurs
+:: Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
-: Compare genome to find targets for point mutation
+:: Add 4.92 g of CsCl to 6 ml of phage suspension buffer to create a 1.6 g/ml density gradient.
-: Playing around with phage genome
+: Layer two centrifuge tubes with 2 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 3 mL of 1.3 g/mL.
-:: Knocking out the polymerase
+:Layer T4 and T7 on top of the gradient in separate tubes.
-:: Change genome size - experiment – could go bigger or smaller
+:Fill the remaining space in the tube with phage suspension buffer to 3-5 mL from the top.
-: T7 specific
+:Centrifuge at 26500 rpms (100,000 g) for 2.5 hours.
-:: Play around with minor and major capsid protein – CCC site
+:Leave overnight in the refrigerator.
+'''V) Results'''
+: After centrifugation, we were able to see a distinct band of phage in both the T4 and T7 gradients.  The T7 band was significantly lower in the tube than the T4 band.  Next time we run the procedure, we will be running the T7 phage through a higher concentrated gradient.  After leaving the gradients overnight in the refrigerator, the gradients mixed together and we had difficulty seeing the distinct bands to extract the phage.  We will have to rerun the experiment.
 </font>