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7.17 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
- phage suspension buffer
IV) Actual Procedure
- Create different concentrations of CsCl solutions to create a gradient.
- Add 1.6391 g of CsCl to 4 mL of phage suspension buffer to create a 1.3 g/ml density gradient.
- Add 4.0953 g of CsCl to 6 mL of phage suspension buffer to create a 1.5 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.
- 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.
- For Dialysis, the aliquot we chose to dialyzed was placed in dialysis tubing in 500x the concentration of phage in phage suspension buffer. Since our samples were 2 mL, this meant 1,000 mL
phage suspension buffer. The samples were left spinning in the buffer for 30 min before transferring it to a new bottle of 1,000 mL PSB. this was repeated 3 times.
V) Results
- The mutant phage had the strongest band that we have ever seen at the wild type location between 1.5019 and 1.5025 There was also a smaller band at 1.5003. These results are very exciting and infer that there should be a large amount of mutant phage.
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