Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/6.17CsClGradient

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Phage Purification May - June Notebook: Experiments

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 6.12 PEG Purification

CsCl

phage suspension buffer

IV) Actual Procedure

Create different concentrations of CsCl solutions to create a gradient.

Add 1.64 g of CsCl to 4 ml of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 4.10 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.

Add 5.76 g of CsCl to 6 ml of phage suspension buffer to create a 1.7 g/ml density gradient.

Layer two centrifuge tubes with 3 mL 1.7 g/mL, 3 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 2 mL of 1.3 g/mL.

Layer T4 and T7 on top of the gradient in separate tubes(as much as is available).

Fill the remaining space in the tube with phage suspension buffer to the top.

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

Extract using a needle and puncturing the side of the tube, placing the needle underneath the band.

V) Results

After centrifugation,