"
Team:Exeter/lab book/1gp/wk6
From 2012.igem.org
| Line 113: | Line 113: | ||
<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:# | + | <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:# | + | <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:# | + | <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> | ||
| Line 122: | Line 122: | ||
<p><u><b>Wednesday 15th August (11.00)</u></b></p> | <p><u><b>Wednesday 15th August (11.00)</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:# | + | <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> | ||
| Line 129: | Line 129: | ||
<p><u><b>Thursday 16th August (13.00)</u></b></p> | <p><u><b>Thursday 16th August (13.00)</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:# | + | <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:# | + | <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:# | + | <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> | ||
Revision as of 10:55, 26 September 2012
Follow our Blog
Follow us on Twitter
|
|
|
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. |
|
