Team:HokkaidoU Japan/Notebook/H Stem/Construction

From 2014.igem.org

Revision as of 09:12, 14 October 2014 by Osamu (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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!