Team:Tianjin/Note/6803

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Team Tianjin-Attribution

Week3(8/29/2016-9/4/2016)

  • pMV-G19
  • pMV-G15
  • Colonies were used to inoculate overnight cultures.
  • Plasmids were isolated using a miniprep kit.
    • Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli
  • 19 amplification at 65.0°C with 19.rev/fwd primes
    •         PCR worked, positive control worked, no amplification of 19
  • 15 amplification at 65.0°C with 15.rev/fwd primes
    • PCR worked, positive control worked, no amplification of 15
  • A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.
  • This result was confirmed by sequencing.
  • The fragments of 19 and 15 were purified with PCR Purification Kit.
    • Show More Ligation of 15 with 19
  • Fragment of 19 was phosphorylated and then purified with DNA Purification Kit.
  • 19 was ligated into T vectors via TA clone and transformed into E.coli via heat shock.
  • A colony PCR was performed with five colonies
    • Gel electrophoresis showed that 5 colonies were positive for insertion of 19. Two of these colonies containing 19 were used to inoculate overnight cultures.
  • 15 gene fragment was phosphorylated.
  • Plasmids containing T vector with 19(pT-19)were isolated using a miniprep kit.
  • Mono-restriction digest of pT-19 with stu I
  • The enzyme-digested product was dephosphorylation.
  • Dephosphorylated plasmid and phosphorylated gene 15 were connected.
    • Ligation product was transformed into E.coli via heat shock.
  • A colony PCR was performed with twelve colonies.
    • Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment. These two colonies were used to inoculate overnight cultures.
  • Plasmids with correct sequence of 19-15 were isolated using a miniprep kit.
  • Mono-restriction digest of pT-19-15 with Nru I
  • The enzyme-digested product was dephosphorylation.
    • Show More IMG_2956

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    Week4(9/5/2016-9/11/2016)

  • Colonies containing gene 13 were used to inoculate overnight cultures.
  • Plasmids were isolated using a miniprep kit.
    • Amplification of 13 with Q5 High-Fidelity DNA Polymerase out of E.coli
  • 13 amplification at 65.0°C with 13.rev/fwd primes
    • PCR worked, positive control worked, no amplification of 13
  • The fragments of 13 were purified with PCR Purification Kit.
  • 13 gene fragment was phosphorylated.
    • Show More Insertion of Ni promoter and ligation of 13-19-15
  • Ni inducible promoter was ligated into Pcpc 3301 vector.
  • Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
  • Single colonies were obtained by plating.
  • A colony PCR of Ni inducible promoter was performed with 12 colonies.
    • Two of the successful ones were used to inoculate overnight cultures.
  • A colony PCR of pT-13-19-15 was performed with 7 colonies.
    • Two of the successful ones were used to inoculate overnight cultures.
  • Two kinds of plasmids were isolated using a miniprep kit.
    • Show More IMG_2956

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    火影忍者疾风传

    终于来到喀纳斯——中国最西北的角落，此次西北之行可到达的最远处。在这个纬度高达 48 度的地方，山坡上的植被更加茂密；太阳下落得很慢，阳光以一个非常低的角度横扫过来，跨过群山，透过树木，在地上都投出长长的影子，非常好看。

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    火影忍者十分钟疾风传

    告别禾木乡，继续向喀纳斯方向前行，途中经过冲乎尔乡。这里是一个天然牧场，坐落在小盆地，群山环抱，乡民主要以放牧为生。果然，不时就能看见牧羊人赶着几百头“阿勒泰大尾羊”在公路上浩浩荡荡地走着，一路扬起滚滚烟尘，甚是壮观。
    IMG_2861

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    北疆树林

    继续在铁热克提附近沿着蜿蜒的小道行摄，不知不觉间便绕到这座高山的另一侧。这里的杨树长得更高耸茂密，西斜的阳光恰到好处地将树梢照得金黄透亮，一道道树影投射在绒毛地毯般的甸上，构成近乎完美的光影，令人心旷神怡，不舍离。
    IMG_2696

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    Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release.     — 2016 iGEM Team Tianjin