Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP

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Notebook
Lab Documents

anti-sense mRFP Construction

  • Start

  • Primer Annealing

    asB0034_F Tm: 68.7℃ asB0034_R Tm: 68.7℃

  • Digestion

    Cut pHN1257 with NcoI, XhoI(using 10×cut smart)

  • Gel extraction

    digested pHN1257

  • Ethanol Precipitation

    digested pHN1257

  • Ligation

    Ligate pHN1257 with asB0034

  • Transformation

    Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0µL DNA to DH5α 42℃ 1min on ice 2min

  • Colony PCR

    Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1℃ asB0034_R Tm:68.7℃

  • Culture

    Ptrc-stem-asB0034-stem

  • mini-Prep

    Ptrc-stem-asB0034-stem

  • Transformation

    Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice 30min each of 2.5µL DNA to DH5α 42℃ 30sec on ice 2min

  • Complete!

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