Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/8.7CsClGradient

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Phage Purification July - August Notebook: Experiments

8.7 CsCl Gradient

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

T7 mutant phage

CsCl

dialysis tubing

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 2.3466 g of CsCl to 4 mL of phage suspension buffer to create a 1.43 g/ml density gradient.

Add 3.6840 g of CsCl to 6 mL of phage suspension buffer to create a 1.45 g/ml density gradient.

Add 3.8483 g of CsCl to 6 mL of phage suspension buffer to create a 1.47 g/ml density gradient.

Add 4.0978 g of CsCl to 6 mL of phage suspension buffer to create a 1.5003 g/ml density gradient.

Add 2.7362 g of CsCl to 4 mL of phage suspension buffer to create a 1.5011 g/ml density gradient.

Add 2.7406 g of CsCl to 4 mL of phage suspension buffer to create a 1.5019 g/ml density gradient.

Add 4.1159 g of CsCl to 6 mL of phage suspension buffer to create a 1.5025 g/ml density gradient.

Add 2.7489 g of CsCl to 4 mL of phage suspension buffer to create a 1.5034 g/ml density gradient.

Add 4.1283 g of CsCl to 6 mL of phage suspension buffer to create a 1.5040 g/ml density gradient.

Add 4.9222 g of CsCl to 6 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

Add 5.7549 g of CsCl to 6 mL of phage suspension buffer to create a 1.7 g/ml density gradient.

Layer the gradient into two centrifuge tubes, using half of the total volume created for each tube. For example, add 2 mL of 1.3 density to one tube and the other 2 mL to another tube.

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.

V) Results

The phage banded ll at 1.3, allowing for no separation between wild type and mutant. The gradient will have to be run again. No phage was extracted or dialyzed.