Team:HokkaidoU Japan/Notebook/Pre experiment/asmRFP

From 2014.igem.org

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R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451; KOD plus Neo
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R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2&#8451; & as_mRFP_NcoI_R Tm:76.0&#8451;  
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KOD plus Neo
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart) Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
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Cut pHN1257 with NcoI, XhoI (using 10&times;Cut Smart)  
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Cut asmRFP with NcoI, XhoI (using 10&times;Cut Smart)
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asmRFP(on pHN1257) 5&micro;L to DH5&alpha; Turbo
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asmRFP(on pHN1257)  
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5&micro;L to DH5&alpha; Turbo
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asmRFP(on pHN1257) pHN1257_up_F Tm:60.1&#8451; & pHN1257_terminator_R Tm:71.4&#8451; Kapa-Taq
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asmRFP(on pHN1257) pHN1257_up_F Tm:60.1&#8451; & pHN1257_terminator_R Tm:71.4&#8451;  
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Kapa-Taq
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Revision as of 03:55, 18 October 2014

Notebook
Lab Documents

Anti-Sense mRFP Construction

  • Start

  • Get anti-sense vector

    Great thanks to N. Nakashima

  • PCR & PCR purification

    R0040-B0034-E1010-B0015 with as_RBS_XhoI_F Tm:68.2℃ & as_mRFP_NcoI_R Tm:76.0℃
    KOD plus Neo

  • Ethanol Precipitation

    asmRFP

  • Digestion

    Cut pHN1257 with NcoI, XhoI (using 10×Cut Smart)
    Cut asmRFP with NcoI, XhoI (using 10×Cut Smart)

  • Gel Extraction

    pHN1257 asmRFP

  • Ethanol Precipitation

    asmRFP

  • Dephosphorylation

    asmRFP

  • Ligation

    Ligate asmRFP with pHN1257

  • Transformation

    asmRFP(on pHN1257)
    5µL to DH5α Turbo

  • Colony PCR

    asmRFP(on pHN1257) pHN1257_up_F Tm:60.1℃ & pHN1257_terminator_R Tm:71.4℃
    Kapa-Taq

  • Liquid Culture

    asmRFP(on pHN1257)

  • Mini-Prep

    asmRFP(pHN1257)

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/asmRFP"