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<div class="attribution">Week2(8/15/2016-8/21/2016)</div>
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<div class="attribution">Week3(8/29/2016-9/4/2016)</div>
 
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<li>Colonies were used to inoculate overnight cultures.</li>
 
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<b>Amplification of <i>19</i> and <i>15</i> with Q5 High-Fidelity DNA Polymerase out of E.coli </b><br />
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<b>Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli </b><br/>
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Revision as of 14:07, 28 September 2016

TEAM TIANJIN

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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.
    • 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.

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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