Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602011"

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<h3>K1602003 - cis-aconitate decarboxylase (cadA)</h3>
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<h3>K1602011 - 2-Dehydro-3-deoxy-D-pentonate aldolases yagE</h3>
  
  
 
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<figcaption><br><b>Figure 1</b> PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
 
<figcaption><br><b>Figure 1</b> PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
 
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The <em>cadA</em>-gene was synthesized by GeneArt and amplified through PCR using the cadA (FW) and cadA (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis and subsequently purified. The <em>cadA </em>amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. The ligation product was transformed into <em>E.coli </em>Top 10 by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by Sanger sequencing.
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The <i>yagE</i> gene was isolated from <i>Escherichia coli</i> and amplified by colony PCR using the oligonucleotides "yagE FW" and "yagE REV". The PCR product was verified by agarose gel electrophoresis, digested with DpnI for 120 minutes and subsequently purified. The <i>yagE</i> amplicon was digested with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. <i>E.Coli</i> Top10 has been transformed with the ligation product by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones and verified by sanger sequencing.
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Revision as of 15:03, 17 September 2015

K1602011 - 2-Dehydro-3-deoxy-D-pentonate aldolases yagE



Figure 1 PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).
The yagE gene was isolated from Escherichia coli and amplified by colony PCR using the oligonucleotides "yagE FW" and "yagE REV". The PCR product was verified by agarose gel electrophoresis, digested with DpnI for 120 minutes and subsequently purified. The yagE amplicon was digested with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. E.Coli Top10 has been transformed with the ligation product by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones and verified by sanger sequencing.