IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/08

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Gel Extraction, New Digests, and Tentative Plans

  • The Mcherry plate posed a few problems with enzymes, but the cuts had been made properly. It showed that the P site does not cut properly and usually results in partial digestions.
  • Made a new game plan for getting a Kanamyacin cell with three plasmids, RBS, TES, and MCherry.
  • Did a gel extraction of MCherry with X and P sites cut and froze that. Also cut out the MCherry with E and S and X and S sites but stored those with gel, and didn't finish extraction (to be used later if necessary).
  • Attached below is the plan, and the "Tuesday" part of the plan was completed as well.
  • Tuesday:

Digest RBS at S and P (at 3pm) 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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