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

From 2014.igem.org

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P<sub>tet</sub>-B0034-mRFP-dT(6A1) SpeI-PstI-F& P<sub>tet</sub>-SpeI-R(vector), XbaI-B0032-mRFP-F& PstI-SpeI-R(insert), KOD Fx Neo
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P<sub>tet</sub>-B0034-mRFP-dT(6A1) SpeI_PstI_F& P<sub>tet</sub>_SpeI_R(vector), XbaI-B0032-mRFP-F& PstI-SpeI-R(insert), KOD Fx Neo
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Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&deg;C 35min 65&deg;C 10min
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Antactic phosphorylate insert (using 10&times;Antarctic phosphatase buffer) 37&#8451; 35min 65&#8451; 10min
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ligation products on ice  30min each of 5.0&micro;L DNA to DH5&alpha;  42&deg;C       1min on ice 2min
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ligation products on ice  30min each of 5.0&micro;L DNA to DH5&alpha;  42&#8451;        1min on ice 2min
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P<sub>tet</sub>-B0034-mRFP-dT on pSB6A1 EcoI-XbaI-F Tm:70.9 rbs-SpeI-R Tm:59.2
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P<sub>tet</sub>-B0034-mRFP-dT on pSB6A1 EcoI_XbaI_F Tm:70.9&#8451; rbs_SpeI_R Tm:59.2&#8451;
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Revision as of 13:31, 17 October 2014

hogehoge

Ptet-B0032-mRFP-dT

  • Start

  • PCR

    Ptet-B0034-mRFP-dT(6A1) SpeI_PstI_F& Ptet_SpeI_R(vector), XbaI-B0032-mRFP-F& PstI-SpeI-R(insert), KOD Fx Neo

  • Gel extraction

    digested PCR productions

  • Digestion

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

  • Antarctic phosphatase

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

  • Ligation

    Ligate vector with insert

  • Transformation

    ligation products on ice 30min each of 5.0µL DNA to DH5α 42℃ 1min on ice 2min

  • Colony PCR

    Ptet-B0034-mRFP-dT on pSB6A1 EcoI_XbaI_F Tm:70.9℃ rbs_SpeI_R Tm:59.2℃

  • Culture

    Ptet-B0034-mRFP-dT on pSB6A1

  • mini-Prep

    Ptet-B0034-mRFP-dT on pSB6A1

  • Complete!

Retrieved from "http://2014.igem.org/Team:HokkaidoU_Japan/Notebook/Pre_experiment/140910_Ptet-B0032-mRFP-dT"