Team:HokkaidoU Japan/Notebook/H Stem/Construction

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H-Stem Construction

  • Start

  • mini-Prep

    1C3 Vector

  • PCR

    Plac (plasmid) with 100up-F & 200dn-R, Ptet-B0034-mRFP-dT (plasmid) with XhoI-RBS & NcoI-mRFP (anti-sense), double-Terminator (plasmid) with 100up & 200dn

  • Digestion

    Plac with SpeI, anti-sense with NcoI, anti-sense with XhoI, dT with XbaI

  • Primer Annealing

    stem-upstream-f & stem-upstream-r, stem-downstream-f & stem-downstream-r

  • Ligation

    Plac & stem-up & as-up, as-dn & stem-dn & dT

  • PCR

    Plac-stem-as with 100up-F & XhoI-RBS, as-stem-dT with NcoI-mRFP & 100dn

  • PCR

    Plac-stem-as & as-stem-dT with 100up-F & 200dn-R

  • Digestion

    Plac-stem-as-stem-dT with EcoRI & PstI, 1C3 with EcoRI & PstI

  • Ligation

    Plac-stem-as-stem-dT & 1C3

  • Transformation

    Plac-stem-as-stem-dT on 1C3

  • Colony PCR

    Plac-stem-as-stem-dT on 1C3 with stem-up-f & stem-d-r / 100up-F & 200dn-R / EX-F & PS-R

  • Complete!

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