Team:BYU Provo/Notebook/Phage Purification/Summerexp/Period1/Exp/7.17 T4 CsCl Gradient

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Phage Purification July - August Notebook: Experiments

7.17 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

T4 mutant phage

CsCl

phage suspension buffer

IV) Actual Procedure

Create different concentrations of CsCl solutions to create a gradient.

Add 1.6391 g of CsCl to 4 mL of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 4.0953 g of CsCl to 6 mL of phage suspension buffer to create a 1.5 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.

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.

Extract using a needle and puncturing the side of the tube, placing the needle at the bottom of the tube.

Extract about 2 mL of phage at a time. Add about 1.5 to one eppendorf.

When the needle is removed place another eppendorf under the hole to catch any leaking phage and fill it with the remainder of the phage in the needle.

For Dialysis, the aliquot we chose to dialyzed was placed in dialysis tubing in 500x the concentration of phage in phage suspension buffer. Since our samples were 2 mL, this meant 1,000 mL

phage suspension buffer. The samples were left spinning in the buffer for 30 min before transferring it to a new bottle of 1,000 mL PSB. this was repeated 3 times. V) Results

The mutant phage had the strongest band that we have ever seen at the wild type location between 1.5019 and 1.5025 There was also a smaller band at 1.5003. These results are very exciting and infer that there should be a large amount of mutant phage.