Team:HokkaidoU Japan/Notebook/Length/Future Work

From 2014.igem.org

Notebook
Lab Documents

Future Work

  • Start

  • prePCR

    R0040-B0034-E1010-B0015 with Ptet_XhoI_F Tm:75.1℃ & mRFP_400dn_R Tm:67.0℃ KOD plus Neo

  • PCR adding Klenow

    R0040-B0034-E1010-B0015t 20cycles, 35cycles annealing 40℃

  • Gel extraction

    inserts

  • Ethanol precipitation

    inserts

  • Digestion

    Cut inserts and pHN1257 with NcoI, XhoI(using 10×CutSmart)

  • Gel extraction

    inserts, pHN1257

  • Ethanol precipitation

    inserts, pHN1257

  • Dephosphorylation

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

  • Ligation

    Ligate inserts with pHN1257

  • Transformation

    ligation products 5.0µL DNA to DH5α

  • Colony PCR

    sequenses are unknown because of randomizing(on pHN1257) pHN1257_up_F Tm:60.1&#8451 & as_RBS_XhoI_R Tm:68.2℃ Kapa-Taq

  • Complete!

We could not get hoped result though we got some colonies. However, stem region has a problem that PCR is not performed well. It is possible that the constructs were complete.

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