Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/8.26 T7 CsCl Gradient

Phage Purification

March-April

May-June

July-August

September-October

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.

V) Results

The phage banded at 1.5003, which is where wild type may band. There was no lower band, but we extracted and purified anyways and then let the large phage group decide what to do.