Difference between revisions of "Team:Tianjin/Note/6803"

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    火影忍者十分钟疾风传。了解其中最佳CP从小就是青梅竹马。没有月光，除了远处县城依稀的灯光和偶尔驶过的车灯外，便是一片漆黑，伸手不见五指，只能在满天繁星的映衬下依稀看见些随风而动的树影。而这，正是拍摄银河的最佳时机。<br />从小就是青梅竹马。没有月光，除了远处县城依稀的灯光和偶尔驶过的车灯外，便是一片漆黑，伸手不见五指，只能在满天繁星的映衬下依稀看见些随风而动的树影。而这，正是拍摄银河的最佳时机。<br />从小就是青梅竹马。没有月光，除了远处县城依稀的灯光和偶尔驶过的车灯外，便是一片漆黑，伸手不见五指，只能在满天繁星的映衬下依稀看见些随风而动的树影。而这，正是拍摄银河的最佳时机。<br />从小就是青梅竹马。没有月光，除了远处县城依稀的灯光和偶尔驶过的车灯外，便是一片漆黑，伸手不见五指，只能在满天繁星的映衬下依稀看见些随风而动的树影。而这，正是拍摄银河的最佳时机。<br /></p>
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<li><i>19</i> amplification at 65.0°C with 19.rev/fwd primes</li>
 +
<li>PCR worked, positive control worked, no amplification of <i>19</i></li>
 +
<li><i>15</i> amplification at 65.0°C with 15.rev/fwd primes</li>
 +
<li>PCR worked, positive control worked, no amplification of <i>15</i></li>
 +
<li>A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.</li>
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<li>This result was confirmed by sequencing.</li>
 +
<li>The fragments of <i>19</i> and <i>15</i> were purified with PCR Purification Kit.</li>
 +
<b>Ligation of <i>15</i> with <i>19</i></b><br/>
 +
<li>Fragment of <i>19</i> was phosphorylated and then purified with DNA Purification Kit.</li>
 +
<li><i>19</i> was ligated into T vectors via TA clone and transformed into E.coli via heat shock.</li>
 +
<li>A colony PCR was performed with five colonies</li>
 +
<li>Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.</li>
 +
<li>Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.</li>
 +
<li><i>15</i> gene fragment was phosphorylated.</li>
 +
<li>Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.</li>
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<li>Mono-restriction digest of pT-19 with stu I </li>
 +
<li>The enzyme-digested product was dephosphorylation.</li>
 +
<li>Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.</li>
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<li>Ligation product was transformed into E.coli via heat shock.</li>
 +
<li>A colony PCR was performed with twelve colonies.</li>
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<li>Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.</li>
 +
<li>These two colonies were used to inoculate overnight cultures.</li>
 +
<li>Plasmids with correct sequence of  <i>19-15</i>  were isolated using a miniprep kit.</li>
 +
<li>Mono-restriction digest of pT-19-15 with Nru I</li>
 +
<li>The enzyme-digested product was dephosphorylation.</li>
 +
 
 
              
 
              
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             </div>
 
             </div>
 
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Revision as of 14:13, 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.
    • 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。

    查看本辑全部作品 ›

    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