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

Line 158: Line 158:
  
  
−
<li><i>19</i> amplification at 65.0°C with 19.rev/fwd primes</li>
+
<li><i>19</i> amplification at 65.0°C with 19.rev/fwd primes.</li>
−
   &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR worked, positive control worked, no amplification of <i>19</i>
+
   PCR worked, positive control worked, no amplification of <i>19</i>.
−
<li><i>15</i> amplification at 65.0°C with 15.rev/fwd primes</li>
+
<li><i>15</i> amplification at 65.0°C with 15.rev/fwd primes.</li>
−
   PCR worked, positive control worked, no amplification of <i>15</i>
+
   PCR worked, positive control worked, no amplification of <i>15</i>.
 
<li>A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.</li>
 
<li>A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.</li>
 
<li>This result was confirmed by sequencing.</li>
 
<li>This result was confirmed by sequencing.</li>
Line 179: Line 179:
 
<li>Fragment of <i>19</i> was phosphorylated and then purified with DNA Purification Kit.</li>
 
<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><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>A colony PCR was performed with five colonies.</li>
 
   Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.</li>
 
   Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.</li>
 
   Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.</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><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>
 
<li>Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.</li>
−
<li>Mono-restriction digest of pT-19 with stu I </li>
+
<li>Mono-restriction digest of pT-19 with stu I. </li>
 
<li>The enzyme-digested product was dephosphorylation.</li>
 
<li>The enzyme-digested product was dephosphorylation.</li>
 
<li>Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.</li>
 
<li>Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.</li>
Line 192: Line 192:
 
   These two colonies were used to inoculate overnight cultures.</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>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>Mono-restriction digest of pT-19-15 with Nru I.</li>
 
<li>The enzyme-digested product was dephosphorylation.</li>
 
<li>The enzyme-digested product was dephosphorylation.</li>
  
Line 255: Line 255:
  
  
−
<li>Ni inducible promoter was ligated into Pcpc 3301 vector.</li>
+
<li>Ni inducible promoter was ligated into pCPC-3301 vector.</li>
 
<li>Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
 
<li>Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
 
<li>Single colonies were obtained by plating.</li>
 
<li>Single colonies were obtained by plating.</li>
−
<li>A colony PCR of Ni inducible promoter was performed with 12 colonies.</li>
+
<li>A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.</li>
 
Two of the successful ones were used to inoculate overnight cultures.</li>
 
Two of the successful ones were used to inoculate overnight cultures.</li>
 
<li>A colony PCR of pT-13-19-15 was performed with 7 colonies.</li>
 
<li>A colony PCR of pT-13-19-15 was performed with 7 colonies.</li>
Line 310: Line 310:
 
<li>Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
 
<li>Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
 
1.13_fwd and 13_rev on pT-13-19-15<br/>
 
1.13_fwd and 13_rev on pT-13-19-15<br/>
−
2.19_ fwd and 19_rev on pT-13-19-15<br/>
+
2.19_fwd and 19_rev on pT-13-19-15<br/>
−
3. 15_ fwd and 15_rev on pT-13-19-15<br/>
+
3.15_fwd and 15_rev on pT-13-19-15<br/>
−
4. 13_ fwd and 19_rev on pT-13-19-15<br/>
+
4.13_fwd and 19_rev on pT-13-19-15<br/>
−
5. 19_ fwd and 15_rev on pT-13-19-15<br/>
+
5.19_fwd and 15_rev on pT-13-19-15<br/>
−
6. 13_ fwd and 15_rev on pT-13-19-15<br/>
+
6.13_ wd and 15_rev on pT-13-19-15<br/>
 
     <b>The fourth and sixth ones were not successful.</b><br/>
 
     <b>The fourth and sixth ones were not successful.</b><br/>
−
<li><b>Repetition:</b>  Phosphorylated 13 was ligated into T-19-15 and transformed into E.coli via heat shock.</li>
+
<li><b>Repetition:</b>  Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
 
<li><b>Repetition:</b> Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
 
<li><b>Repetition:</b> Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
−
1. 1.13_fwd and 13_rev on pT-13-19-15<br/>
+
1.13_fwd and 13_rev on pT-13-19-15<br/>
−
2. 13_ fwd and 19_rev on pT-13-19-15<br/>
+
2.13_fwd and 19_rev on pT-13-19-15<br/>
 
<b>The second one was failed.</b>
 
<b>The second one was failed.</b>
  
Line 363: Line 363:
 
         <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
         <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
 
<header class="entry-header">
 
<header class="entry-header">
−
<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">火影忍者疾风传</a></h1>
+
<h1 class="entry-title"><a href="https://www.camarts.cn/archives/4252.html" title="查看《火影忍者疾风传》中的全部作品" rel="bookmark">Week6(9/19/2016-9/25/2016)</a></h1>
 
</header><!-- .entry-header -->
 
</header><!-- .entry-header -->
  
 
<div class="entry-content">
 
<div class="entry-content">
−
<p>火影忍者十分钟疾风传。了解其中最佳CP<a href="https://www.camarts.cn/archives/tag/%e5%b8%83%e5%b0%94%e6%b4%a5" title="查看所有与鸣樱相关的照片">鸣樱</a>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>
 +
<li>Medium preparation :BG-11.</li>
 +
<li>19_ fwd and 15_rev were used to amplify <i>19-15</i>.</li>
 +
<li>Gel electrophoresis showed that amplification of fragments was successfull.</li>
 +
<li>Ligated <i>13</i> and <i>19-15</i> via overlap PCR.</li>
 +
    <li>This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.</li>
 +
<li>Restriction digest on pCPC-3031-Ni with Sac I.</li>
 +
<li>The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.</li>
 +
<li>A colony PCR of pT-13-19-15 was performed with 12 colonies.</li>
 +
Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.</li>
 +
<li><i>13-19-15</i> gene fragment was phosphorylated.</li>
 +
<li>Phosphorylated <i>13-19-15</i> was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.</li>
 +
 
 +
<li>Plasmids pT-13-19-15 were isolated using a miniprep kit.</li>
 +
<li>A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.</li>
 +
Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.</li>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<br />
 +
 
 +
 
 
<a href="https://www.camarts.cn/archives/4252.html"><img class="aligncenter size-full wp-image-4256" src="http://static.camarts.cn/images/spinner-1600.gif"  alt="IMG_2956" width="800" height="533" srcset="images/img_1.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /><noscript><img class="aligncenter size-full wp-image-4256" src="http://img.camarts.cn/2016/01/IMG_2956.jpg?imageView/2/w/800/h/800/q/90" alt="IMG_2956" width="800" height="533" srcset="http://img.camarts.cn/2016/01/IMG_2956.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /></noscript></a></p>
 
<a href="https://www.camarts.cn/archives/4252.html"><img class="aligncenter size-full wp-image-4256" src="http://static.camarts.cn/images/spinner-1600.gif"  alt="IMG_2956" width="800" height="533" srcset="images/img_1.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /><noscript><img class="aligncenter size-full wp-image-4256" src="http://img.camarts.cn/2016/01/IMG_2956.jpg?imageView/2/w/800/h/800/q/90" alt="IMG_2956" width="800" height="533" srcset="http://img.camarts.cn/2016/01/IMG_2956.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /></noscript></a></p>
 
<p> <a href="https://www.camarts.cn/archives/4252.html" class="more-link">查看Team Tianjin全部实验 <span class="meta-nav">&rsaquo;</span></a></p>
 
<p> <a href="https://www.camarts.cn/archives/4252.html" class="more-link">查看Team Tianjin全部实验 <span class="meta-nav">&rsaquo;</span></a></p>

Revision as of 15:50, 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.
    • 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

    查看Team Tianjin全部实验 ›

    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(pCPC-3301-Ni) 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

    查看Team Tianjin全部实验 ›

    Week5(9/12/2016-9/18/2016)

  • Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
    • The sequencing results for both of them were error.
  • Colonies containing Ni inducible promoter were used to inoculate overnight cultures.
  • PCR was performed to check if the gene fragments were ligated correctly.
    • 13_ fwd and 15_rev on pT-13-19-15
    Gel electrophoresis showed that it failed.
  • Plasmids pCPC-3031-Ni were isolated using a miniprep kit.
  • Several PCRs were performed to check if the gene fragments were ligated correctly.
    • 1.13_fwd and 13_rev on pT-13-19-15
    2.19_fwd and 19_rev on pT-13-19-15
    3.15_fwd and 15_rev on pT-13-19-15
    4.13_fwd and 19_rev on pT-13-19-15
    5.19_fwd and 15_rev on pT-13-19-15
    6.13_ wd and 15_rev on pT-13-19-15
    The fourth and sixth ones were not successful.
  • Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
  • Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
    • 1.13_fwd and 13_rev on pT-13-19-15
    2.13_fwd and 19_rev on pT-13-19-15
    The second one was failed.
    IMG_2956

    查看Team Tianjin全部实验 ›

    Week6(9/19/2016-9/25/2016)

  • Medium preparation :BG-11.
  • 19_ fwd and 15_rev were used to amplify 19-15.
  • Gel electrophoresis showed that amplification of fragments was successfull.
  • Ligated 13 and 19-15 via overlap PCR.
  • This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.
  • Restriction digest on pCPC-3031-Ni with Sac I.
  • The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.
  • A colony PCR of pT-13-19-15 was performed with 12 colonies.
    • Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.
  • 13-19-15 gene fragment was phosphorylated.
  • Phosphorylated 13-19-15 was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.
  • Plasmids pT-13-19-15 were isolated using a miniprep kit.
  • A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.
    • Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.
    IMG_2956

    查看Team Tianjin全部实验 ›

    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

    查看本辑全部作品 ›

    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

    查看本辑全部作品 ›

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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