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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
- Filling the tube all the way to the top of the base of the neck made it easier to pull the tubes out avoiding any accidental mixing of gradients. We noticed only one band in the tubes. The one band that we observed seemed to have bacterial debris just from a naked eye observation, we will be spot testing the band to see if it contained any phage.
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