|
- Phage Purification
- March-April
- May-June
- July-August
- September-October
|
9.7 CsCl Gradient
I) Purpose
- Try to figure out which density the WT T7 phage bands with our new CsCl stock.
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
- We didn't observe any banding in either of the centrifuge tubes. We are still trying to figure out why it refuses to band now, but has in past experiments.
|
"
