Team:Exeter/lab book/gibs/wk5

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      <!--Project Division Links-->
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/proto"; style="color:#57b947">Protocols</a>
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        &nbsp;|&nbsp;
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      &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Novel Polysaccharide Production</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
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        &nbsp;|&nbsp;
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      &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Gibson Assembly</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/novpol/wk1"; style="color:#57b947">Showcasing Polysaccharide Production</a>
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        &nbsp;|&nbsp;
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      &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk1"; style="color:#57b947">Single Gene Plasmids and Enzyme Characterisation</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk1"; style="color:#57b947">The 3-Gene Inducible Plasmid</a>
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        <p>
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      <p>  
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       <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#57b947">Operon Construction</a>     
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        </p>
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       &nbsp;|&nbsp;
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      <a href="https://2012.igem.org/Team:Exeter/lab_book/glyco/wk1"; style="color:#57b947">Glycobase</a>
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    <!--Project Division Week Hyperlinks-->
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  <!--Project Division Week Hyperlinks-->
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#1d1d1b">9th - 13th July</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"; style="color:#1d1d1b">9th - 13th July</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk6"; style="color:#1d1d1b">13th - 24th August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk6"; style="color:#1d1d1b">13th - 17th August</a>
         <p>
         <p>
         -
         -
         </p>
         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk7"; style="color:#1d1d1b">27th - 31st August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk7"; style="color:#1d1d1b">20th - 24th August</a>
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        <p>
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        -
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        </p>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk8"; style="color:#1d1d1b">27th - 31st August</a>
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        <p>
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        -
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        </p>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk9"; style="color:#1d1d1b">3rd - 7th September</a>
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        <p>
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        -
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        <a href="https://2012.igem.org/Team:Exeter/Results#usepoly"; style="color:#e30614" target="_blank"><font size="3"><b>Results: Part 1</b></font></a>
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        <p>
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        -
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        <a href="https://2012.igem.org/Team:Exeter/Results/GvsB"; style="color:#e30614" target="_blank"><font size="3"><b>Results: Part 2</b></font></a>
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       <p><b><u>Gibson Assembly: 6th - 10th August 2012</u></b></p>
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       <p><b><u>Operon Construction: 6th - 10th August 2012</u></b></p>
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     <!------------ENTER LAB BOOK HERE: PLEASE INCLUDE DATES WITHIN THE WEEK------------>  
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     <!<p><b>**Monday 6.8.12**</b></p><br>
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<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A BioBrick assembly</u></a> of RBS-<i>wbbC</i>(d) (BBa_B0034 + BBa_K764005)</p><br><p>
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Upstream</p><p>
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RBS 3 (BBa_B0034) plasmid DNA - 8.1 µl</p><p>
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Water - 14.4 µl</p><br><p>
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Downstream <i>wbbC</i>(d) </p><p>
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DNA  - 2.8 µl</p><p>
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Water  - 19.7 µl</p><br><p>
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Destination plasmid pSB1C3 (chloramphenicol resistant) <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#57b947"><u>Digestion</u></a></p><p>
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<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/10" style="color:#57b947"><u>Ligation</u></a></p><br><p>
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Upstream: 2 µl</p><p>
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Downstream: 2 µl</p><p>
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MilliQ water 2.5</p><br><p>
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 +
23 °C (room temp.) incubation for 1 hour</p><p>
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20 minute heat kill 80 °C</p><br><p>
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**Wednesday 8.8.12**</p><br>
 +
<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformed</u></a> RBS-<i>wbbC</i>(d) (BBa_B0034 + BBa_K764005)</p>
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<p>2 µl into 25 µl competent cells</p><p>
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50 µl and 150 µl LB(chlor) spread plates made</p><br><p>
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 +
 
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<b>**Thursday 9.8.12**</b></p><br>
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<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Transferred</u></a> RBS-<i>wbbC</i>(d) (BBa_B0034 + BBa_K764005) transformed colonies to LB(chlor) medium</p><br><p>
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 +
 
 +
<b>**Friday 10.8.12**</b></p><br><p>
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 +
<p> RBS-<i>wbbC</i>(d) transformed cells broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C</p>
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<p>Supernatant discarded</p>
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<p>Miniprep according to <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Fermentas protocol</u></a></p>
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<p><b><i>Step 10</i></b>45µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation</p>
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    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p>
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Latest revision as of 00:10, 27 September 2012

ExiGEM2012 Lab Book Gibs wk5

Operon Construction: 6th - 10th August 2012

 **Monday 6.8.12**


3A BioBrick assembly of RBS-wbbC(d) (BBa_B0034 + BBa_K764005)


Upstream

RBS 3 (BBa_B0034) plasmid DNA - 8.1 µl

Water - 14.4 µl


Downstream wbbC(d)

DNA - 2.8 µl

Water - 19.7 µl


Destination plasmid pSB1C3 (chloramphenicol resistant) Digestion

Ligation


Upstream: 2 µl

Downstream: 2 µl

MilliQ water 2.5


23 °C (room temp.) incubation for 1 hour

20 minute heat kill 80 °C


**Wednesday 8.8.12**


Transformed RBS-wbbC(d) (BBa_B0034 + BBa_K764005)

2 µl into 25 µl competent cells

50 µl and 150 µl LB(chlor) spread plates made


**Thursday 9.8.12**


Transferred RBS-wbbC(d) (BBa_B0034 + BBa_K764005) transformed colonies to LB(chlor) medium


**Friday 10.8.12**


RBS-wbbC(d) transformed cells broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C

Supernatant discarded

Miniprep according to Fermentas protocol

Step 1045µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation

Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley   |   Contact Us   |   Site Map

Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/gibs/wk5"