Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/8.12CsClGradient

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Latest revision as of 00:36, 28 September 2013

Phage Purification July - August Notebook: Experiments

Phage Purification

8.12 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.

1.1 - 2 mL

1.2 - 2 mL

1.25 - 2 mL

1.3 - 2 mL

1.35 - 2mL

1.4 - 2 mL

1.5 - 3 mL

1.6 - 2 mL

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.

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

Place phage in dialysis tubing and place in flask with 1 L of phage suspension buffer for 30 minutes at 4^◦ C.

Repeat previous step three more times.

Remove purified phage from dialysis tubing and store in 4^◦ C.

V) Results

The Phage did not band anywhere

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 : Create different concentrations of CsCl solutions to create a gradient.
-:: Add 0.5500 g of CsCl to 4 mL of phage suspension buffer to create a 1.2 g/ml density gradient.
+:: 1.1 - 2 mL
-:: Add 1.6175 g of CsCl to 4 mL of phage suspension buffer to create a 1.296 g/ml density gradient.
+::1.2 - 2 mL
-:: Add 1.6207 g of CsCl to 4 mL of phage suspension buffer to create a 1.2966 g/ml density gradient.
+::1.25 - 2 mL
-:: Add 0.8128 g of CsCl to 6 mL of phage suspension buffer to create a 1.2975 g/ml density gradient.
+::1.3 - 2 mL
-:: Add 1.6288 g of CsCl to 6 mL of phage suspension buffer to create a 1.2981 g/ml density gradient.
+::1.35 - 2mL
-:: Add 1.6332 g of CsCl to 6 mL of phage suspension buffer to create a 1.2989 g/ml density gradient.
+::1.4 - 2 mL
-:: Add 1.6375 g of CsCl to 4 mL of phage suspension buffer to create a 1.2997 g/ml density gradient.
+::1.5 - 3 mL
-:: Add 1.6408 g of CsCl to 4 mL of phage suspension buffer to create a 1.3003 g/ml density gradient.
+::1.6 - 2 mL
-:: Add 1.6451 g of CsCl to 6 mL of phage suspension buffer to create a 1.3011 g/ml density gradient.
-:: Add 1.6494 g of CsCl to 4 mL of phage suspension buffer to create a 1.3019 g/ml density gradient.
-:: Add 1.6527 g of CsCl to 6 mL of phage suspension buffer to create a 1.3025 g/ml density gradient.
-:: Add 0.8288 g of CsCl to 6 mL of phage suspension buffer to create a 1.3034 g/ml density gradient.
-:: Add 1.6608 g of CsCl to 6 mL of phage suspension buffer to create a 1.3040 g/ml density gradient.
-:: Add 3.2815 g of CsCl to 6 mL of phage suspension buffer to create a 1.6 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.