Team:HokkaidoU Japan/Notebook/Pre experiment/140910 Ptet-B0032-mRFP-dT

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P-tet-B0032-mRFP-pdT

  • Start

  • PCR

    Ptet-B0034-mRFP-dT(6A1) SpeI-PstI-F& Ptet-SpeI-R(vector), XbaI-B0032-mRFP-F& PstI-SpeI-R(insert), KOD Fx Neo

  • Gel extraction

    digested PCR productions

  • Digestion

    Cut vector with SpeI, PstI (using 10×H buffer) Cut insert with XbaI, PstI (using 10×M buffer)

  • Antarctic phosphatase

    Antactic phosphorylate insert (using 10×Antarctic phosphatase buffer) 37°C 35min 65°C 10min

  • Ligation

    Ligate vector with insert

  • Transformation

    ligation products on ice 30min each of 5.0µL DNA to DH5α 42°C 1min on ice 2min

  • Colony PCR

    Ptet-B0034-mRFP-dT on pSB6A1 EcoI-XbaI-F Tm:70.9 rbs-SpeI-R Tm:59.2

  • Culture

    Ptet-B0034-mRFP-dT on pSB6A1

  • mini-Prep

    Ptet-B0034-mRFP-dT on pSB6A1

  • Complete!

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