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6.3 CsCl Gradient Phage Purification
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 and T7 purified phage from 5.26 PEG Purification
- CsCl
- phage suspension buffer
IV) Actual Procedure
- Create different concentrations of CsCl solutions to create a gradient.
- Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
- Add 4.1 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.
- Layer two centrifuge tubes with 2 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 3 mL of 1.3 g/mL.
- Layer T4 and T7 on top of the gradient in separate tubes.
- Fill the remaining space in the tube with phage suspension buffer to 3-5 mL from the top.
- Centrifuge at 26500 rpms (100,000 g) for 2.5 hours.
- Leave overnight in the refrigerator.
V) Results
- After centrifugation, we were able to see a distinct band of phage in both the T4 and T7 gradients. The T7 band was significantly lower in the tube than the T4 band. Next time we run the procedure, we will be running the T7 phage through a higher concentrated gradient. After leaving the gradients overnight in the refrigerator, the gradients mixed together and we had difficulty seeing the distinct bands to extract the phage. We will have to rerun the experiment.
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