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- Phage Purification
- March-April
- May-June
- July-August
- September-October
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9.11 CsCl Gradient
I) Purpose
- Try to figure out which density the WT T7 phage bands with our new CsCl stock and new Phage Suspension Buffer.
II) Expected Outcome
- We should see a band of WT T7 phage around the 1.2 - 1.5 density.
III) Reagants Used
- T7 mutant phage
- CsCl
- phage suspension buffer
IV) Actual Procedure
- Create 2 tubes of different concentrations of CsCl solutions to create a gradient.
- For the first tube
- Add 0.4225 g of CsCl to 3 mL of phage suspension buffer to create a 1.1 g/ml density gradient.
- Add 0.8245 g of CsCl to 3 mL of phage suspension buffer to create a 1.2 g/ml density gradient.
- Add 1.2293 g of CsCl to 3 mL of phage suspension buffer to create a 1.3 g/ml density gradient.
- Add 1.6371 g of CsCl to 3 mL of phage suspension buffer to create a 1.4 g/ml density gradient.
- Add 2.0477 g of CsCl to 3 mL of phage suspension buffer to create a 1.5 g/ml density gradient.
- Add 2.4611 g of CsCl to 3 mL of phage suspension buffer to create a 1.6 g/ml density gradient.
- Add 2.8774 g of CsCl to 3 mL of phage suspension buffer to create a 1.7 g/ml density gradient.
- Add 3.2966 g of CsCl to 3 mL of phage suspension buffer to create a 1.8 g/ml density gradient.
- For the second tube
- Add 0.5497 g of CsCl to 2 mL of phage suspension buffer to create a 1.2 g/ml density gradient.
- Add 0.6844 g of CsCl to 2 mL of phage suspension buffer to create a 1.25 g/ml density gradient.
- Add 1.2293 g of CsCl to 3 mL of phage suspension buffer to create a 1.3 g/ml density gradient.
- Add 1.4328 g of CsCl to 3 mL of phage suspension buffer to create a 1.35 g/ml density gradient.
- Add 1.0914 g of CsCl to 2 mL of phage suspension buffer to create a 1.4 g/ml density gradient.
- Add 1.8420 g of CsCl to 3 mL of phage suspension buffer to create a 1.45 g/ml density gradient.
- Add 2.0477 g of CsCl to 3 mL of phage suspension buffer to create a 1.5 g/ml density gradient.
- Add 1.6407 g of CsCl to 2 mL of phage suspension buffer to create a 1.6 g/ml density gradient.
- Add 1.9183 g of CsCl to 2 mL of phage suspension buffer to create a 1.7 g/ml density gradient.
- Add 2.1977 g of CsCl to 2 mL of phage suspension buffer to create a 1.8 g/ml density gradient.
- Layer the two gradients into two separate centrifuge tubes.
- 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.
V) Results
- Bands were visible at the 1.3 level in both tubes. However, in the tube that had more differentiation between steps, the band was very faint. this may be why the band disappeared when a more specific gradient was ran. Now that we know where normal T7 bands, we will be able to extractabove and below the band for small and large mutants.
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