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- Phage Purification
- March-April
- May-June
- July-August
- September-October
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8.14 T4 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
- T4 mutant 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 2.3466 g of CsCl to 4 mL of phage suspension buffer to create a 1.43 g/ml density gradient.
- Add 3.6840 g of CsCl to 6 mL of phage suspension buffer to create a 1.45 g/ml density gradient.
- Add 3.8483 g of CsCl to 6 mL of phage suspension buffer to create a 1.47 g/ml density gradient.
- Add 4.0978 g of CsCl to 6 mL of phage suspension buffer to create a 1.5003 g/ml density gradient.
- Add 2.7362 g of CsCl to 4 mL of phage suspension buffer to create a 1.5011 g/ml density gradient.
- Add 2.7406 g of CsCl to 4 mL of phage suspension buffer to create a 1.5019 g/ml density gradient.
- Add 4.1159 g of CsCl to 6 mL of phage suspension buffer to create a 1.5025 g/ml density gradient.
- Add 2.7489 g of CsCl to 4 mL of phage suspension buffer to create a 1.5034 g/ml density gradient.
- Add 4.1283 g of CsCl to 6 mL of phage suspension buffer to create a 1.5040 g/ml density gradient.
- Add 4.9222 g of CsCl to 6 mL of phage suspension buffer to create a 1.6 g/ml density gradient.
- Add 5.7549 g of CsCl to 6 mL of phage suspension buffer to create a 1.7 g/ml density gradient.
- Layer the gradient into two centrifuge tubes, using half of the total volume created for each tube. For example, add 2 mL of 1.3 density to one tube and the other 2 mL to another tube.
- 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 three more times.
- Remove purified phage from dialysis tubing and store in 4◦ C.
V) Results
- The phage banded at 1.45 (upper Faint Band - possibly small mutated phage) and made a large band between 1.5019 and 1.5025 (possibly mutated large phage closer to 1.5025 with normal phage at 1.5019). The control had a faint band at 1.5019.
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