From 2013.igem.org
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| | '''V) Results''' | | '''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. | + | : |
| - | [[File:T7_1st_Gradient_09.6.13.jpg | thumb|none|alt=A|T7 in 1st gradient]]
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| - | [[File:T7_2nd_Gradient_09.6.13.jpg | thumb|none|alt=A|T7 in 2nd gradient]]
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Revision as of 20:40, 16 September 2013
- Phage Purification September - October Notebook: Experiments
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- Overview
- March-April
- May-June
- July-August
- September-October
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9.16 CsCl Gradient
I) Purpose
- Isolate both small and large mutant T7 phage.
II) Expected Outcome
- We most likely will not see bands but the small phage should be found in the high end of the gradient and large phage will be found in the lower end of the gradient. We will extract the entire gradient and perform spot tests to confirm.
III) Reagants Used
- T7 mutant phage
- CsCl
- phage suspension buffer
IV) Actual Procedure
- Create 3 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 1.2131 g of CsCl to 3 mL of phage suspension buffer to create a 1.2960 g/ml density gradient.
- Add 1.2155 g of CsCl to 3 mL of phage suspension buffer to create a 1.2966 g/ml density gradient.
- Add 1.2192 g of CsCl to 3 mL of phage suspension buffer to create a 1.2975 g/ml density gradient.
- Add 1.2216 g of CsCl to 3 mL of phage suspension buffer to create a 1.2981 g/ml density gradient.
- Add 1.2249 g of CsCl to 3 mL of phage suspension buffer to create a 1.2989 g/ml density gradient.
- Add 1.2281 g of CsCl to 3 mL of phage suspension buffer to create a 1.2997 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 1.6407 g of CsCl to 2 mL of phage suspension buffer to create a 1.6 g/ml density gradient.
- For the third tube
- Add 0.8196 g of CsCl to 2 mL of phage suspension buffer to create a 1.3 g/ml density gradient.
- Add 1.2306 g of CsCl to 3 mL of phage suspension buffer to create a 1.3003 g/ml density gradient.
- Add 1.2338 g of CsCl to 3 mL of phage suspension buffer to create a 1.3011 g/ml density gradient.
- Add 1.2371 g of CsCl to 3 mL of phage suspension buffer to create a 1.3019 g/ml density gradient.
- Add 1.2395 g of CsCl to 3 mL of phage suspension buffer to create a 1.3025 g/ml density gradient.
- Add 1.2432 g of CsCl to 3 mL of phage suspension buffer to create a 1.3034 g/ml density gradient.
- Add 1.2456 g of CsCl to 3 mL of phage suspension buffer to create a 1.3040 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 1.3651 g of CsCl to 2 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.
- Layer the three gradients into three separate centrifuge tubes.
- Add 4 mL of mutant and wild type phage to the top of the gradient in all tubes. The wild type phage is layered on the first gradient. The mutant phage are layered on the second and third gradients
- 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
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