Team:MIT/pdestmcherry

I NEEDED A PAGE!!!!!!!

During the insertion of the the pENTR constructs into the pDEST, the mCherry gets cut out of the plasmid. When transformed into eColi on plates containing Ampicillin, the mCherry acts as a negative screening tool for choosing colonies that contain the properly-assembled plasmid of choice. the pDEST plasmid here is designed with the express purpose of distribution for use by other teams and scientists.

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Using the pDEST with mCherry is extremely similar to normal ligation reaction, transformations, and plating protocols.

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Many thanks are owed to the Tufts iGEM 2016 team, for agreeing to test our construct for us!

Materials
  • Promoter pENTR plasmid: L4-Promoter-R1 working concentration 5 fmol/μL
  • Gene pENTR plasmid: L1-Gene-L2 working concentration 5 fmol/μL
  • pDEST:mCherry plasmid working concentration 10 fmol/μL
  • Nuclease-free TE
  • 200μL strip tube for each reaction
  • 5x LR Clonase II
  • Proteinase K

    • Procedure

    Into each tube: </li> SUBLISTS?????????

  • 1 µl of the promoter pENTR
  • 1 µl of the gene pENTR
  • 1 µl of the pDEST-mCherry

    • Add 1 µl of TE </li> Add 1 µl of LR clonase (kept in -80)</li> Cap tubes, flick to mix and pulse-spin to collect liquid at the bottom</li> Incubate between 12-24 hours at room temperature. </li>

    Proteinase K-kill

    Add 1 μL proteinase K to each tube Flick tubes to mix and pulse-spin Incubate at 37° for 15 minutes, or room temperature for an hour. Can be used in transformation or stored at -20° indefinitely