Team:BYU Provo/Notebook/Phage Purification/Fallexp/Period1/Exp/9.16CsClGradient

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Phage Purification September - October Notebook: Experiments

9.16 CsCl Gradient

I) Purpose

Isolate both small and large mutant T7 phage.

II) Expected Outcome

We most likely will not see bands but the small phage should be found in the high end of the gradient and large phage will be found in the lower end of the gradient. We will extract the entire gradient and perform spot tests to confirm.

III) Reagants Used

T7 mutant phage

CsCl

phage suspension buffer

IV) Actual Procedure

Create 3 tubes of different concentrations of CsCl solutions to create a gradient.

For the first tube

Add 0.4225 g of CsCl to 3 mL of phage suspension buffer to create a 1.1 g/ml density gradient.

Add 0.8245 g of CsCl to 3 mL of phage suspension buffer to create a 1.2 g/ml density gradient.

Add 1.2293 g of CsCl to 3 mL of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 1.6371 g of CsCl to 3 mL of phage suspension buffer to create a 1.4 g/ml density gradient.

Add 2.0477 g of CsCl to 3 mL of phage suspension buffer to create a 1.5 g/ml density gradient.

Add 2.4611 g of CsCl to 2.5 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

Add 2.8774 g of CsCl to 2 mL of phage suspension buffer to create a 1.7 g/ml density gradient.

Add 3.2966 g of CsCl to 2 mL of phage suspension buffer to create a 1.8 g/ml density gradient.

For the second tube

Add 0.5497 g of CsCl to 2 mL of phage suspension buffer to create a 1.2 g/ml density gradient.

Add 1.2131 g of CsCl to 3 mL of phage suspension buffer to create a 1.2960 g/ml density gradient.

Add 1.2155 g of CsCl to 3 mL of phage suspension buffer to create a 1.2966 g/ml density gradient.

Add 1.2192 g of CsCl to 3 mL of phage suspension buffer to create a 1.2975 g/ml density gradient.

Add 1.2216 g of CsCl to 3 mL of phage suspension buffer to create a 1.2981 g/ml density gradient.

Add 1.2249 g of CsCl to 3 mL of phage suspension buffer to create a 1.2989 g/ml density gradient.

Add 1.2281 g of CsCl to 3 mL of phage suspension buffer to create a 1.2997 g/ml density gradient.

Add 1.2293 g of CsCl to 3 mL of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 1.6371 g of CsCl to 3 mL of phage suspension buffer to create a 1.4 g/ml density gradient.

Add 1.6407 g of CsCl to 2 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

For the third tube

Add 0.8196 g of CsCl to 2 mL of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 1.2306 g of CsCl to 3 mL of phage suspension buffer to create a 1.3003 g/ml density gradient.

Add 1.2338 g of CsCl to 3 mL of phage suspension buffer to create a 1.3011 g/ml density gradient.

Add 1.2371 g of CsCl to 3 mL of phage suspension buffer to create a 1.3019 g/ml density gradient.

Add 1.2395 g of CsCl to 3 mL of phage suspension buffer to create a 1.3025 g/ml density gradient.

Add 1.2432 g of CsCl to 3 mL of phage suspension buffer to create a 1.3034 g/ml density gradient.

Add 1.2456 g of CsCl to 3 mL of phage suspension buffer to create a 1.3040 g/ml density gradient.

Add 1.0914 g of CsCl to 2 mL of phage suspension buffer to create a 1.4 g/ml density gradient.

Add 1.3651 g of CsCl to 2 mL of phage suspension buffer to create a 1.5 g/ml density gradient.

Add 1.6407 g of CsCl to 2 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

Layer the three gradients into three separate centrifuge tubes.

Add 4 mL of mutant and wild type phage to the top of the gradient in all tubes. The wild type phage is layered on the first gradient. The mutant phage are layered on the second and third gradients

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 by poking a small hole in the bottom of the centrifuge tube and let the gradient drip into eppendorf tubes (2 mL in each tube).

V) Results

No bands showed up on the mutant phage gradients. This is what we expected. The small phage team will be spot testing each aliquot to determine where the phage are and see if there are any mutants.