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- Phage Purification
- March-April
- May-June
- July-August
- September-October
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8.16 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 mutant phage
- CsCl
- phage suspension buffer
IV) Actual Procedure
- Create different concentrations of CsCl solutions to create a gradient.
- Add 0.5497 g of CsCl to 2 mL of phage suspension buffer to create a 1.2 g/ml density gradient.
- Add 1.6175 g of CsCl to 4 mL of phage suspension buffer to create a 1.2960 g/ml density gradient.
- Add 1.6207 g of CsCl to 4 mL of phage suspension buffer to create a 1.2966 g/ml density gradient.
- Add 0.8128 g of CsCl to 2 mL of phage suspension buffer to create a 1.2975 g/ml density gradient.
- Add 1.6288 g of CsCl to 4 mL of phage suspension buffer to create a 1.2981 g/ml density gradient.
- Add 1.6332 g of CsCl to 4 mL of phage suspension buffer to create a 1.2989 g/ml density gradient.
- Add 1.6375 g of CsCl to 4 mL of phage suspension buffer to create a 1.2997 g/ml density gradient.
- Add 1.6408 g of CsCl to 4 mL of phage suspension buffer to create a 1.3003 g/ml density gradient.
- Add 1.6451 g of CsCl to 4 mL of phage suspension buffer to create a 1.3011 g/ml density gradient.
- Add 1.6494 g of CsCl to 4 mL of phage suspension buffer to create a 1.3019 g/ml density gradient.
- Add 1.6527 g of CsCl to 4 mL of phage suspension buffer to create a 1.3025 g/ml density gradient.
- Add 0.8288 g of CsCl to 2 mL of phage suspension buffer to create a 1.3034 g/ml density gradient.
- Add 1.6608 g of CsCl to 4 mL of phage suspension buffer to create a 1.3040 g/ml density gradient.
- Add 1.6407 g of CsCl to 4 mL of phage suspension buffer to create a 1.6 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.
- Add 4 mL of mutant phage to the top of the gradient in both tubes
- 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 at the bottom of the tube.
- Extract about 2 mL of phage at a time. Add about 1.5 to one eppendorf.
- When the needle is removed place another eppendorf under the hole to catch any leaking phage and fill it with the remainder of the phage in the needle.
V) Results
- The phage didn't band anywhere. After running a spot test it appears that the phage where present in every aliquot extracted.
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