Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/5.26 PEG Purification

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Phage Purification May - June Notebook: Experiments

5.26 PEG Purification

I) Purpose

The first step in the phage purification process. Purify phage from bacterial debris.

II) Expected Outcome

Phage will be purified from the bacterial debris to be further purified in a CsCl gradient.

III) Reagants Used

phage suspension buffer

10 mM Tris-HCl

100 mM NaCl

10 mM MgCl₂

DNase I

RNase A

chloroform

NaCl powder

PEG 8000

T4 and T7 bacterial lysate

IV) Actual Procedure

Add DNase and RNase to T4 and T7 bacterial lysates.

Add chloroform to complete lysis and let the preparation sit for 30 minutes at room temperature.

Dissolve solid NaCl and let cool at 4^◦ C for 1 h.

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 were unable to see the distinct bands to extract the phage. We will have to rerun the experiment.

@@ Line 43: / Line 43: @@
 :
 : phage suspension buffer
-::10 mM Tris-HCl
+:: 10 mM Tris-HCl
-::100 mM NaCl
+:: 100 mM NaCl
 :: 10 mM MgCl<sub>2</sub>
 : DNase I
-:RNase A
+: RNase A
-:NaCl powder
+: chloroform
-:PEG 8000
+: NaCl powder
+: PEG 8000
+: T4 and T7 bacterial lysate
 '''IV) Actual Procedure'''
-: Create different concentrations of CsCl solutions to create a gradient.
+: Add DNase and RNase to T4 and T7 bacterial lysates.
-:: Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
+: Add chloroform to complete lysis and let the preparation sit for 30 minutes at room temperature.
-:: Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
+: Dissolve solid NaCl and let cool at 4<sup>◦</sup> C for 1 h.
-:: 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 for 2.5 hours.
-:Leave overnight in the refrigerator.
 '''V) Results'''