Team:HokkaidoU Japan/Notebook/H Stem/Construction

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Notebook
Lab Documents

H-Stem Construction

  • Start

  • Mini-Prep

    pSB1C3 vector

  • PCR

    R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
    R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃
    B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

  • Digestion

    R0010 with SpeI
    anti-sense with NcoI, XhoI
    B0015 with XbaI

  • Primer Annealing

    stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃
    stem_downstream_F Tm:71.4℃ & stem_downstream_R Tm:71.4℃

  • Ligation

    R0010 & stem-up & as-up
    as-dn & stem-dn & B0015

  • PCR

    R0010-stem-as with 100up_F Tm:62.6℃ & XhoI_RBS_R Tm:68.2℃
    as-stem-B0015 with NcoI-mRFP_F Tm:76.0℃ & 200dn Tm:66.6℃

  • PCR

    R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

  • Digestion

    Cut R0010-stem-as-stem-B0015 with EcoRI & PstI
    Cut pSB1C3 with EcoRI & PstI

  • Ligation

    Ligate R0010-stem-as-stem-B0015 with pSB1C3

  • Transformation

    R0010-stem-as-stem-B0015(on pSB1C3)
    5.0µL to DH5alpha;

  • Colony PCR

    R0010-stem-as-stem-B0015(on pSB1C3) with
    stem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃
    100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
    EX_F Tm:70.4℃ & PS_R Tm:74.6℃

  • Complete!

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