Team:HokkaidoU Japan/Notebook/Pre experiment/Stem replacement

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Notebook
Lab Documents

Stem Region Replacement

  • Start

  • PCR

    pHN1257 stem_F Tm:70.6℃ terminator_R Tm:71.4℃ KOD FX NEO

  • Transformation

    R0010 on ice 30min each of 5.0µL DNA to DH5α 42℃ 1min on ice 2min

  • Digestion

    Cut R0010 with PstI, SpeI (using 10xH buffer) and pHN1257 with XbaI, PstI (using 10xM buffer)

  • Ligation

    Ligate R0010 with pHN1257

  • Transformation

    Ligation products on ice 30min each of 5.0µL DNA to DH5α 42℃ 1min on ice 2min

  • Colony PCR

    R0010-stem-dT 100up_F pHN1257terminator_R

  • mini-Prep

    R0010-stem-dT

  • PCR(confirm)

    R0010-stem-dT 100up_F pHN1257terminator_R KOD FX NEO

  • Complete!

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