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

Overview

March-April

May-June

July-August

September-October

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

NaCl powder

PEG 8000

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