Team:HokkaidoU Japan/Notebook/asB0034/asE1010 1A3 Construction

From 2014.igem.org

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R0010-stem-asE1010-stem-B0015(on pSB1C3) EcoRI_XbaI_F Tm:74.5&#8451; & Ptet_SpeI_R(insert) Tm:72.6&#8451; KOD Fx Neo
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R0010-stem-asE1010-stem-B0015(on pSB1C3) EcoRI_XbaI_F Tm:74.5&#8451; & Ptet_SpeI_R(insert) Tm:72.6&#8451;  
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KOD Fx Neo
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Cut  vector with EcoRI, PstI (using 10&times;H buffer) Cut  insert with EcoRI, PstI (using 10&times;M buffer)
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Cut  vector with EcoRI, PstI (using 10&times;H buffer)  
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Cut  insert with EcoRI, PstI (using 10&times;M buffer)
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R0010-stem-asE1010-stem-B0015(on pSB1A3) 5.0&micro;L to DH5&alpha;
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R0010-stem-asE1010-stem-B0015(on pSB1A3)  
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5.0&micro;L to DH5&alpha;
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R0010-stem-asE1010-stem-B0015(on pSB1A3) EcoI_XbaI_F Tm:74.5&#8451; & PstI_SpeI_R Tm:74.6&#8451; Kapa-Taq
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R0010-stem-asE1010-stem-B0015(on pSB1A3) EcoI_XbaI_F Tm:74.5&#8451; & PstI_SpeI_R Tm:74.6&#8451;
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Kapa-Taq
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Revision as of 03:05, 18 October 2014

Notebook
Lab Documents

Anti-Sense E1010 on 1A3 Construction

  • Start

  • PCR

    R0010-stem-asE1010-stem-B0015(on pSB1C3) EcoRI_XbaI_F Tm:74.5℃ & Ptet_SpeI_R(insert) Tm:72.6℃
    KOD Fx Neo

  • Digestion

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

  • Dephosphorylation

    Antactic phosphorylate pSB1A3 (using 10×Antarctic phosphatase buffer) 37°C 35min 65℃ 10min

  • Ligation

    Ligate pSB1A3 with R0010-stem-asE1010-stem-B0015

  • Transformation

    R0010-stem-asE1010-stem-B0015(on pSB1A3)
    5.0µL to DH5α

  • Colony PCR

    R0010-stem-asE1010-stem-B0015(on pSB1A3) EcoI_XbaI_F Tm:74.5℃ & PstI_SpeI_R Tm:74.6℃
    Kapa-Taq

  • Culture

    R0010-stem-asE1010-stem-B0015(on pSB1A3)

  • Mini-Prep

    R0010-stem-asE1010-stem-B0015(on pSB1A3)

  • Complete!

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