IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/08: Difference between revisions
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*Tuesday: | *Tuesday: | ||
Digest | Digest RBS at S and P and run overnight. Already have Tes cut at X and P. | ||
Later | Later | ||
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Ligate: TesA part into RBS backbone part | Ligate: TesA part into RBS backbone part | ||
E| | E|| RBS | S| X| TESA | P|| P (ready to cut at ends) | ||
Transform: Transform the plasmids into cells (mix plasmids with competent cells) | Transform: Transform the plasmids into cells (mix plasmids with competent cells) | ||
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Plate Cells | Plate Cells | ||
Pick colonies to make seeds/tubes | Pick colonies to make seeds/tubes | ||
Dr. Nielsons Suggestions | Miniprep seed cultures, and gel extract with complete plasmid | ||
Digest P and S sites at end of Tes, and Ligate MCherry at X and P sites | |||
Gel extract longest piece (all together RBS, TES, and MCherry) | |||
E| RBS | S| X| TESA | S| X| MCherry | P | |||
Dr. Nielsons Further Suggestions (in case this procedure doesn't work) | |||
1. Two plasmids | 1. Two plasmids |
Revision as of 15:02, 8 July 2014
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Gel Extraction, New Digests, and Tentative Plans
Digest RBS at S and P and run overnight. Already have Tes cut at X and P. Later Gel: Run in gel and extract TesA part without backbone and RBS linear plasmid with backbone. Ligate: TesA part into RBS backbone part E|| RBS | S| X| TESA | P|| P (ready to cut at ends) Transform: Transform the plasmids into cells (mix plasmids with competent cells) Both plasmids into cell Plate Cells Pick colonies to make seeds/tubes Miniprep seed cultures, and gel extract with complete plasmid Digest P and S sites at end of Tes, and Ligate MCherry at X and P sites Gel extract longest piece (all together RBS, TES, and MCherry) E| RBS | S| X| TESA | S| X| MCherry | P Dr. Nielsons Further Suggestions (in case this procedure doesn't work) 1. Two plasmids 2. Attach primers to TesA and MC 3. Order Tes with RBS attached 4. Start ACL with Wax 5. Might not add MCherry to plasmid, instead add to chromosome to make permanent change later. 6. Use PCR to complete some of steps
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