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Revision as of 20:37, 1 July 2013
| Phage Purification May - June Notebook: Experiments
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- Overview
- 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
- 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.
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 extracted phage from this band and will be performing a titer on it next class.
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