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6.17 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 6.12 PEG Purification
- CsCl
- 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 two centrifuge tubes 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 T4 and 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.
V) Results
- After centrifugation,
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