Team:BYU Provo/Notebook/Phage Purification/Fallexp/Period1/Exp/9.76CsClGradient

Phage Purification

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Try to figure out which density the WT T7 phage bands with our new CsCl stock.

II) Expected Outcome

We should see a band of WT T7 phage around the 1.2 - 1.5 density.

III) Reagants Used

T7 mutant phage

CsCl

phage suspension buffer

IV) Actual Procedure

Create 2 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 3 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

Add 2.8774 g of CsCl to 3 mL of phage suspension buffer to create a 1.7 g/ml density gradient.

Add 3.2966 g of CsCl to 3 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 0.6844 g of CsCl to 2 mL of phage suspension buffer to create a 1.25 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 1.4328 g of CsCl to 3 mL of phage suspension buffer to create a 1.35 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 1.8420 g of CsCl to 3 mL of phage suspension buffer to create a 1.45 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 1.6407 g of CsCl to 2 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

Add 1.9183 g of CsCl to 2 mL of phage suspension buffer to create a 1.7 g/ml density gradient.

Add 2.1977 g of CsCl to 2 mL of phage suspension buffer to create a 1.8 g/ml density gradient.

Layer the two gradients into two separate centrifuge tubes.

Add 4 mL of mutant phage to the top of the gradient in both tubes

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

We didn't observe any banding in either of the centrifuge tubes. We are still trying to figure out why it refuses to band now, but has in past experiments.

T7 in 1st gradient

T7 in 2nd gradient