Team:HokkaidoU Japan/Notebook/asB0034/asB0034-construction

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      </ul>
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      <div class="step-recipe">
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asB0034_F Tm: 68.7&#8451; asB0034_R Tm: 68.7&#8451;
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asB0034_F Tm:68.7&#8451; asB0034_R Tm:68.7&#8451;
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      </ul>
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Ptrc-stem-asB0034-stem(pHN1257) on ice 30min each of 5.0&micro;L DNA to DH5&alpha; 42&#8451;        1min on ice 2min
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Ptrc-stem-asB0034-stem on pHN1257  
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5.0&micro;L DNA to DH5&alpha;
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Ptrc-stem-asB0034-stem(pHN1257) pHN1257_up_F Tm:60.1&#8451; asB0034_R Tm:68.7&#8451;
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Ptrc-stem-asB0034-stem(on pHN1257) pHN1257_up_F Tm:60.1&#8451; & asB0034-R Tm:68.7&#8451;
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Ptrc-stem-asB0034-stem
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Ptrc-stem-asB0034-stem(on pHN1257)
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mini-Prep
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Mini-Prep
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Ptrc-stem-asB0034-stem
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Ptrc-stem-asB0034-stem(on pHN1257)
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Transformation
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Double Transformation
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Ptrc-stem-asB0034-stem on pHN1257&Ptet-B0034-mRFP-dt on pSB6A1 on ice  30min each of 2.5&micro;L DNA to DH5&alpha; 42&#8451;    30sec on ice  2min
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Ptrc-stem-asB0034-stem(on pHN1257) & R0040-B0034-E1010-B0015(on pSB6A1)
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                                      each of 2.5&micro;L DNA to DH5&alpha;  
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Latest revision as of 15:24, 9 September 2015

Notebook
Lab Documents

asB0034 Construction

  • Start

  • Primer Annealing

    asB0034_F Tm:68.7℃ asB0034_R Tm:68.7℃

  • Digestion

    Cut pHN1257 with NcoI, XhoI(using 10×cut smart)

  • Gel extraction

    digested pHN1257

  • Ethanol Precipetation

    digested pHN1257

  • Ligation

    Ligate pHN1257 with asB0034

  • Transformation

    Ptrc-stem-asB0034-stem on pHN1257
    5.0µL DNA to DH5α

  • Colony PCR

    Ptrc-stem-asB0034-stem(on pHN1257) pHN1257_up_F Tm:60.1℃ & asB0034-R Tm:68.7℃

  • Culture

    Ptrc-stem-asB0034-stem(on pHN1257)

  • Mini-Prep

    Ptrc-stem-asB0034-stem(on pHN1257)

  • Double Transformation

    Ptrc-stem-asB0034-stem(on pHN1257) & R0040-B0034-E1010-B0015(on pSB6A1)
    each of 2.5µL DNA to DH5α

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/asB0034/asB0034-construction"