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

From 2013.igem.org


Phage Purification September - October Notebook: Experiments



Phage Purification
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9.7 CsCl Gradient


I) Purpose

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.
A
T7 in 1st gradient
A
T7 in 2nd gradient