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

Line 302: Line 302:
  
 
<li>Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.</li>
 
<li>Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.</li>
The sequencing results for both of them were error.
+
<b>The sequencing results for both of them were error.</b>
 
<li>Colonies containing Ni inducible promoter were used to inoculate overnight cultures.</li>
 
<li>Colonies containing Ni inducible promoter were used to inoculate overnight cultures.</li>
 
<li>PCR was performed to check if the gene fragments were ligated correctly.</li>
 
<li>PCR was performed to check if the gene fragments were ligated correctly.</li>
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<div class="note-content3">
 
 
 
 
<li><i>13</i> amplification at 65.0°C with 13.rev/fwd primes</li>
 
PCR worked, positive control worked, no amplification of <i>13</i></li>
 
<li>The fragments of <i>13</i> were purified with PCR Purification Kit.</li>
 
<li><i>13</i> gene fragment was phosphorylated.</li>
 
         
 
       
 
 
</div>
 
<a class="expand-btn3">Show More</a>
 
 
 
<b>Insertion of Ni promoter and ligation of <i>13-19-15</i></b><br/>
 
 
<div class="note-content4">
 
 
 
<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>Single colonies were obtained by plating.</li>
 
<li>A colony PCR of Ni inducible promoter was performed with 12 colonies.</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>
 
  Two of the successful ones were used to inoculate overnight cultures.</li>
 
<li>Two kinds of plasmids were isolated using a miniprep kit.</li>
 
 
 
         
 
       
 
</div>
 
            <a class="expand-btn4">Show More</a>
 
  
  

Revision as of 15:37, 28 September 2016

TEAM TIANJIN


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

    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_ fwd and 15_rev on pT-13-19-15
    The fourth and sixth ones were not successful.
  • Repetition: Phosphorylated 13 was ligated into T-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. 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


    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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    info_outline
    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