Team:Exeter/lab book/1gp/wk6

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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/1gp/wk8"; style="color:#1d1d1b">27th - 31st August</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk11"; style="color:#1d1d1b">17th - 21st September</a>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk9"; style="color:#1d1d1b">3rd - 7th September</a>
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            <p>
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        <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk10"; style="color:#1d1d1b">10th - 14th September</a>
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         <p>
         <p>
         -
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk11"; style="color:#1d1d1b">17th - 21st September</a>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk12"; style="color:#1d1d1b">24th - 28th September</a>
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         -
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/1gp/wk12"; style="color:#1d1d1b">24th - 28th September</a></font>
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         <a href="https://2012.igem.org/Team:Exeter/Results/characterise"; style="color:#e30614" target="_blank"><font size="3"><b>Results</b></font></a>
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</font>
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     <p><u><b>Monday 13th August (9.00)</u></b></p>
     <p><u><b>Monday 13th August (9.00)</u></b></p>
-
<p>• PCR amplification of <i>WbbC</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#1d1d1b"><u>3-step PCR</u></a></p>
+
<p>• PCR amplification of <i>WbbC</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3-step PCR</u></a></p>
-
<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Adding cultures</u></a> with ligated WbnK-terminator and WclY-terminator in liquid medium overnight</p>
+
<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Adding cultures</u></a> with ligated WbnK-terminator and WclY-terminator in liquid medium overnight</p>
<p><u><b>Tuesday 14th August (9.00)</u></b></p>
<p><u><b>Tuesday 14th August (9.00)</u></b></p>
-
<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#1d1d1b"><u>Mini-Prepping</u></a> of ligated WbnK-terminator and WclY-terminator</p>
+
<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57b947"><u>Mini-Prepping</u></a> of ligated WbnK-terminator and WclY-terminator</p>
<p>• Gel Electrophoresis to check fragment sizes of ligated WbnK-terminator, WclY-terminator and PCR product generated from yesterday's PCR attempt</p>
<p>• Gel Electrophoresis to check fragment sizes of ligated WbnK-terminator, WclY-terminator and PCR product generated from yesterday's PCR attempt</p>
<p> Gel Electrophoresis showed that WbnK-terminator mini-prep 1 had worked, WbnK-terminator mini-prep 2 and all WclY-terminator mini-preps plus the PCR product were not discovered. For the PCR product, it was thought that the annealing temperature was too high. Lane 1 = DNA hyperladder, Lane 2 = WbnK-terminator mini-prep 1 (expected: 2 bands at 1003bp and 2070bp), Lane 3 = WbnK-terminator mini-prep 2 (expected: 2 bands at 1003bp and 2070bp), Lane 4 = WclY-terminator mini-prep 1 (expected: 2 bands at 1156bp and 2070bp), Lane 5 = WclY-terminator mini-prep 2 (expected: 2 bands at 1156bp and 2070bp), Lane 6 = WbbC PCR attempt, Lane 7 = DNA hyperladder.</p>
<p> Gel Electrophoresis showed that WbnK-terminator mini-prep 1 had worked, WbnK-terminator mini-prep 2 and all WclY-terminator mini-preps plus the PCR product were not discovered. For the PCR product, it was thought that the annealing temperature was too high. Lane 1 = DNA hyperladder, Lane 2 = WbnK-terminator mini-prep 1 (expected: 2 bands at 1003bp and 2070bp), Lane 3 = WbnK-terminator mini-prep 2 (expected: 2 bands at 1003bp and 2070bp), Lane 4 = WclY-terminator mini-prep 1 (expected: 2 bands at 1156bp and 2070bp), Lane 5 = WclY-terminator mini-prep 2 (expected: 2 bands at 1156bp and 2070bp), Lane 6 = WbbC PCR attempt, Lane 7 = DNA hyperladder.</p>
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<p><u><b>Wednesday 15th August (11.00)</u></b></p>
<p><u><b>Wednesday 15th August (11.00)</u></b></p>
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<p>• PCR amplification of <i>WbbC</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#1d1d1b"><u>3-step PCR</u></a></p>
+
<p>• PCR amplification of <i>WbbC</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3-step PCR</u></a></p>
<p>• Send off WbnK-terminator for sequencing</p>
<p>• Send off WbnK-terminator for sequencing</p>
<p>• Gel Electrophoresis to check fragment sizes of PCR product</p>
<p>• Gel Electrophoresis to check fragment sizes of PCR product</p>
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<p><u><b>Thursday 16th August (13.00)</u></b></p>
<p><u><b>Thursday 16th August (13.00)</u></b></p>
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<p>• PCR amplification of <i>WbbC</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#1d1d1b"><u>3-step PCR</u></a></p>
+
<p>• PCR amplification of <i>WbbC</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3-step PCR</u></a></p>
-
<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Adding cultures</u></a> with ligated WclY-terminator in liquid medium overnight</p>
+
<p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Adding cultures</u></a> with ligated WclY-terminator in liquid medium overnight</p>
<p> PCR amplification of WbbC was followed using the same protocol but the annealing temperature was taken down from 65°C to 55°C.</p>
<p> PCR amplification of WbbC was followed using the same protocol but the annealing temperature was taken down from 65°C to 55°C.</p>
<p> Gel Electrophoresis did not show a PCR product and it was thought that there was an issue with the BL21 genomic DNA template.</p>
<p> Gel Electrophoresis did not show a PCR product and it was thought that there was an issue with the BL21 genomic DNA template.</p>
<p><u><b>Friday 17th August (10.00)</u></b></p>
<p><u><b>Friday 17th August (10.00)</u></b></p>
-
<p>• PCR amplification of <i>WbbC</i> and <i>FadR</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#1d1d1b"><u>3-step PCR</u></a></p>
+
<p>• PCR amplification of <i>WbbC</i> and <i>FadR</i> from <i>E.coli</i> BL21(DE3) using <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/12" style="color:#57b947"><u>3-step PCR</u></a></p>
<p> FadR is a transcription factor involved in fatty acid metabolism and was used as a false-positive.</p>
<p> FadR is a transcription factor involved in fatty acid metabolism and was used as a false-positive.</p>
<p> Gel Electrophoresis did not show any PCR products and it was thought that there was an issue with the BL21 genomic DNA template.</p>
<p> Gel Electrophoresis did not show any PCR products and it was thought that there was an issue with the BL21 genomic DNA template.</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 23:54, 26 September 2012

ExiGEM2012 Lab Book 1GP wk6

Single Gene Plasmids and Enzyme Characterisation: 13th - 17th August 2012

Monday 13th August (9.00)

• PCR amplification of WbbC from E.coli BL21(DE3) using 3-step PCR

• Adding cultures with ligated WbnK-terminator and WclY-terminator in liquid medium overnight

Tuesday 14th August (9.00)

• Mini-Prepping of ligated WbnK-terminator and WclY-terminator

• Gel Electrophoresis to check fragment sizes of ligated WbnK-terminator, WclY-terminator and PCR product generated from yesterday's PCR attempt

Gel Electrophoresis showed that WbnK-terminator mini-prep 1 had worked, WbnK-terminator mini-prep 2 and all WclY-terminator mini-preps plus the PCR product were not discovered. For the PCR product, it was thought that the annealing temperature was too high. Lane 1 = DNA hyperladder, Lane 2 = WbnK-terminator mini-prep 1 (expected: 2 bands at 1003bp and 2070bp), Lane 3 = WbnK-terminator mini-prep 2 (expected: 2 bands at 1003bp and 2070bp), Lane 4 = WclY-terminator mini-prep 1 (expected: 2 bands at 1156bp and 2070bp), Lane 5 = WclY-terminator mini-prep 2 (expected: 2 bands at 1156bp and 2070bp), Lane 6 = WbbC PCR attempt, Lane 7 = DNA hyperladder.

Wednesday 15th August (11.00)

• PCR amplification of WbbC from E.coli BL21(DE3) using 3-step PCR

• Send off WbnK-terminator for sequencing

• Gel Electrophoresis to check fragment sizes of PCR product

PCR amplification of WbbC was followed using the same protocol but the annealing temperature was taken down from 65°C to 60°C.

Gel Electrophoresis did not show a PCR product and it was thought that the annealing temperature was too high again.

Thursday 16th August (13.00)

• PCR amplification of WbbC from E.coli BL21(DE3) using 3-step PCR

• Adding cultures with ligated WclY-terminator in liquid medium overnight

PCR amplification of WbbC was followed using the same protocol but the annealing temperature was taken down from 65°C to 55°C.

Gel Electrophoresis did not show a PCR product and it was thought that there was an issue with the BL21 genomic DNA template.

Friday 17th August (10.00)

• PCR amplification of WbbC and FadR from E.coli BL21(DE3) using 3-step PCR

FadR is a transcription factor involved in fatty acid metabolism and was used as a false-positive.

Gel Electrophoresis did not show any PCR products and it was thought that there was an issue with the BL21 genomic DNA template.

No cultures containing ligated WclY-terminator were found.

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/1gp/wk6"