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Team:Exeter/lab book/1gp/wk4
From 2012.igem.org
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<u><b>Monday 30th July (9.00)</u></b> | <u><b>Monday 30th July (9.00)</u></b> | ||
| - | <p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/8" style="color:# | + | <p>• <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/8" style="color:#57b947"><u>Genomic extraction</u></a> from <i>E.coli</i> BL21(DE3)</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/7" 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/7" style="color:#57b947"><u>two-step PCR</u></a></p> |
<p>• Gel Electrophoresis to check fragment size of <i>WbbC</i> PCR product</p> | <p>• Gel Electrophoresis to check fragment size of <i>WbbC</i> PCR product</p> | ||
<p>The following concentration (in ng/µL) obtained for the BL21(DE3) genomic DNA were: | <p>The following concentration (in ng/µL) obtained for the BL21(DE3) genomic DNA were: | ||
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<p><u><b>Tuesday 31st July (15.00)</p></u></b> | <p><u><b>Tuesday 31st July (15.00)</p></u></b> | ||
| - | <p>• <b>IDT re-suspension</b> and <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:# | + | <p>• <b>IDT re-suspension</b> and <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>transformation</u></a> of <i>WbbC</i>(with point mutation)</p> |
<p><u><b>Wednesday 1st August (15.00)</p></u></b> | <p><u><b>Wednesday 1st August (15.00)</p></u></b> | ||
| - | <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 WbbC(with point mutation) plasmids into liquid medium and incubation overnight</p> |
<p> Protocol was followed using ampicillin for WbbC(with point mutation) plasmids as the selection antibiotic.</p> | <p> Protocol was followed using ampicillin for WbbC(with point mutation) plasmids as the selection antibiotic.</p> | ||
<p><u><b>Thursday 2nd August (9.00)</p></u></b> | <p><u><b>Thursday 2nd August (9.00)</p></u></b> | ||
| - | <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 WbbC(with point mutation) plasmid</p> |
<p> • Gel Electrophoresis to check fragment sizes of WbbC(with point mutation) plasmid</p> | <p> • Gel Electrophoresis to check fragment sizes of WbbC(with point mutation) plasmid</p> | ||
<p> The following concentrations (in ng/µL) was obtained:</p> | <p> The following concentrations (in ng/µL) was obtained:</p> | ||
Revision as of 10:52, 26 September 2012
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Single Gene Plasmids and Enzyme Characterisation: 30th July - 3rd August 2012 Monday 30th July (9.00)• Genomic extraction from E.coli BL21(DE3) • PCR amplification of WbbC from E.coli BL21(DE3) using two-step PCR • Gel Electrophoresis to check fragment size of WbbC PCR product The following concentration (in ng/µL) obtained for the BL21(DE3) genomic DNA were: o 1. - 7.8. o 2. - 6.6. Gel Electrophoresis did not show any correct PCR products (expected: 1113bp band) and it was discovered that the reverse primer was incorrect and had actually amplified a shorter section of WbbC, rather than the whole gene. Thus the correct reverse primer was ordered. Lane 1 = DNA hyperladder, Lane 2 = WbbC attempt from genomic DNA template 1, Lane 3 = WbbC attempt from genomic DNA template 2.
Tuesday 31st July (15.00) • IDT re-suspension and transformation of WbbC(with point mutation) Wednesday 1st August (15.00) • Adding cultures with WbbC(with point mutation) plasmids into liquid medium and incubation overnight Protocol was followed using ampicillin for WbbC(with point mutation) plasmids as the selection antibiotic. Thursday 2nd August (9.00) • Mini-Prepping of WbbC(with point mutation) plasmid • Gel Electrophoresis to check fragment sizes of WbbC(with point mutation) plasmid The following concentrations (in ng/µL) was obtained: o WbbC(with point mutation) - 181.6. Gel Electrophoresis showed that WbbC(with point mutation) did not work because whilst there was a band, it was the wrong size (expected: 1113bp). This procedure was repeated again. Lane 1 = DNA hyperladder, Lane 2 = Mini-prepped WbbC.
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