IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/08: Difference between revisions

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4. Start ACL with Wax  
4. Start ACL with Wax  
5. Might not add MCherry to plasmid, instead add to chromosome to make permanent change later.  
5. Might not add MCherry to plasmid, instead add to chromosome to make permanent change later.  
6.    Use PCR to complete some of steps




Revision as of 14:38, 8 July 2014

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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 TesA at X and P sites and RBS at S and P and run overnight

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|X| RBS | S| X| TESA | P| P|

Then cut at E and P sites, gel extract, and ligate with K backbone. Same with MCherry

Transform: Transform the plasmids into cells (mix plasmids with competent cells) Both plasmids into cell

Plate Cells Pick colonies to make seeds/tubes

Dr. Nielsons Suggestions:

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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