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

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R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8&#8451;& Ptet_SpeI_R Tm:72.6&#8451; (vector)
R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8&#8451;& Ptet_SpeI_R Tm:72.6&#8451; (vector)
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        XbaI_B0032_mRFP_F Tm:76.3&#8451; & PstI_SpeI_R Tm:74.6&#8451; (insert)
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        KOD Fx Neo
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XbaI_B0032_mRFP_F Tm:76.3&#8451; & PstI_SpeI_R Tm:74.6&#8451; (insert)
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KOD Fx Neo
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Cut  vector with SpeI, PstI (using 10&times;H buffer)  
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Cut  vector with SpeI, PstI (using 10&times;H buffer)  
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        Cut  insert with XbaI, PstI (using 10&times;M buffer)
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Cut  insert with XbaI, PstI (using 10&times;M buffer)
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R0040-B0032-E1010-B0015(on pSB1A6)
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R0040-B0032-E1010-B0015(on pSB1A6)
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        5.0&micro;L DNA to DH5&alpha;   
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5.0&micro;L DNA to DH5&alpha;   
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Latest revision as of 15:25, 9 September 2015

Notebook
Lab Documents

Ptet-B0032-mRFP-dT

  • Start

  • PCR

    R0040-B0034-E1010-B0015(on pSB6A1) SpeI_PstI_F Tm:72.8℃& Ptet_SpeI_R Tm:72.6℃ (vector)
    XbaI_B0032_mRFP_F Tm:76.3℃ & PstI_SpeI_R Tm:74.6℃ (insert)
    KOD Fx Neo

  • Gel extraction

    PCR products

  • Digestion

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

  • Dephosphorylation

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

  • Ligation

    Ligate vector with insert

  • Transformation

    R0040-B0032-E1010-B0015(on pSB1A6)
    5.0µL DNA to DH5α

  • Colony PCR

    R0040-B0032-E1010-B0015(on pSB6A1) EcoI_XbaI_F Tm:70.9℃ & RBS_SpeI_R Tm:59.2℃

  • Culture

    R0040-B0032-E1010-B0015(on pSB6A1)

  • Mini-Prep

    R0040-B0032-E1010-B0015(on pSB6A1)

  • Complete!

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