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

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Latest revision as of 15:26, 9 September 2015

Notebook
Lab Documents

Stem Region Replacement

  • Start

  • PCR

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

  • Transformation

    R0010
    each of 5.0µL DNA to DH5α

  • Digestion

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

  • Ligation

    Ligate R0010 with pHN1257

  • Transformation

    Ligation products
    each of 5.0µL DNA to DH5α

  • Colony PCR

    R0010-stem-B0015 with 100up_F pHN1257terminator_R

  • Mini-Prep

    R0010-stem-B0015

  • PCR(confirm)

    R0010-stem-dT with 100up_F pHN1257terminator_R
    KOD FX NEO

  • Complete!

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