Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/6.12PEGPurification

Overview

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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

chloroform

NaCl powder

PEG 8000

T4 and T7 bacterial lysate

IV) Actual Procedure

Add 166.5 µL DNase and 22.5 µL RNase to T4 and T7 bacterial lysates.

Add .0904 mL chloroform to complete lysis and let the preparations sit for 30 minutes at room temperature.

Dissolve 1.32 g of solid NaCl and let cool at 4^◦ C for 1 hour.

Centrifuge the suspensions at 6500 rpms (7000 g) for 10 minutes at 4^◦ C. Remove supernatant and put in clean flasks.

Dissolve 3.96 g PEG 8000 and let sit at 4^◦ C for 1 hour.

Centrifuge the suspensions at 8000 rpms (10000 g) for 15 minutes at 4^◦ C. Discard the supernatant.

Turn the centrifuge bottles over to let sit for 5 minutes to drain off any remaining supernatant.

Suspend the pellets in .675 mL phage suspension buffer and let sit overnight at 4^◦ C.

V) Results

We were able to purify phage to a point that it can now be used in the CsCl gradient. We finished with phage dissolved in phage suspension buffer. We will be using this solution in a later class to further purify in a CsCl gradient.