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Team:Duke/Notebook/August
From 2014.igem.org
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<li>9 ul Master Mix added to 1 ul of pdCas9 in one tube and 1 ul of pSB1C3-dCas9-tracrRNA in another tube</li> | <li>9 ul Master Mix added to 1 ul of pdCas9 in one tube and 1 ul of pSB1C3-dCas9-tracrRNA in another tube</li> | ||
| - | <li>Gel | + | <li>Gel results were consistent with an unsuccessful ligation. 3 bands in the experimental column (4kb, 1.8 kb, and 0.9 kb) were expected. The results were more consistent with pSB1C3 and no insert. The band size in the pdCas9 control lane also looked wrong because it was too big. The pdCas9 tube may have been faulty or the BamHI might have been faulty.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
Revision as of 17:08, 11 August 2014
August 8
Objective: Backbone switching
Delta Ghoshal, Matthew Faw
Redo minipreps from yesterday - culture tubes grew overnight from salvaged cells
- pSB1C3-dCas9-tracrRNA - 171.3 ng/ul
- pSB3C5-R0040-a-tracr 1 - 257.5 ng/ul
- pSB3C5-R0040-a-tracr 2 - 202.7 ng/ul
- pSB3C5-R0040-a-tracr 3 - 200.5 ng/ul
- pSB3C5-R0040-a-tracr 4 - 198.3 ng/ul
Analytical digest of Tube 1 (pSB1C3-dCas9-tracrRNA) using XhoI and BamHI
- 2 hour digest at 37 degrees C
- 3X Master Mix:
- 3 ul Cutsmart
- 22.5 ul water
- 0.75 ul XhoI
- 0.75 ul BamHI
- 9 ul Master Mix added to 1 ul of pdCas9 in one tube and 1 ul of pSB1C3-dCas9-tracrRNA in another tube
- Gel results were consistent with an unsuccessful ligation. 3 bands in the experimental column (4kb, 1.8 kb, and 0.9 kb) were expected. The results were more consistent with pSB1C3 and no insert. The band size in the pdCas9 control lane also looked wrong because it was too big. The pdCas9 tube may have been faulty or the BamHI might have been faulty.
