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- Phage Purification
- March-April
- May-June
- July-August
- September-October
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6.24 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 purified phage
- CsCl
- dialysis tubing
- phage suspension buffer
IV) Actual Procedure
- Create different concentrations of CsCl solutions to create a gradient.
- Add 1.64 g of CsCl to 4 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
- Add 4.10 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
- Add 4.92 g of CsCl to 6 ml of phage suspension buffer to create a 1.6 g/ml density gradient.
- Add 5.76 g of CsCl to 6 ml of phage suspension buffer to create a 1.7 g/ml density gradient.
- Layer a centrifuge tube with 3 mL 1.7 g/mL, 3 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 2 mL of 1.3 g/mL.
- Layer T7 on top of the gradient in separate tubes(as much as is available).
- 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 two more times.
- Remove purified phage from dialysis tubing and store in 4◦ C.
V) Results
- We were able to successfully extract phage from the CsCl. Dr. Grose explained that the band which we had previously thought to be the bacterial debris was probably the purified phage. We removed the phage from this band and purified it through dialysis. Next class we will be performing a titer to determine the concentration of the phage.
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