Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602013"

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<h3>K1602003 - cis-aconitate decarboxylase (cadA)</h3>
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<h3>K1602013 - Aldehyde Reductase (YqhD)</h3>
  
  
 
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<img class="transparent" alt="https://static.igem.org/mediawiki/2015/1/12/TU_Darmstadt_PCR_xylB_xylC_yjhG_yagE_yqhD.png" src="https://static.igem.org/mediawiki/2015/1/12/TU_Darmstadt_PCR_xylB_xylC_yjhG_yagE_yqhD.png">
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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>
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<figcaption><br><b>Figure 1</b> PCR of <i>yqhD</i>. The size of the amplified product was around 1,4 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>YqhD</i>-gene was isolated from K12 <i>E. coli</i> and amplified by colony PCR using the <i>YqhD</i> (FW) and <i>YqhD</i> (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with <i>Dpn</i>I for 120 minutes and subsequently purified. The <i>YqhD</i> amplicon was digested with <i>EcoR</i>I and <i>Pst</i>I and ligated into a pSB1C3 vector using T4-ligase. <i>E. coli</i> Top 10 were 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 which were verified by sanger sequencing.
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Latest revision as of 19:43, 18 September 2015

K1602013 - Aldehyde Reductase (YqhD)


https://static.igem.org/mediawiki/2015/1/12/TU_Darmstadt_PCR_xylB_xylC_yjhG_yagE_yqhD.png

Figure 1 PCR of yqhD. The size of the amplified product was around 1,4 kbp. DNA marker: 2-Log DNA Ladder (NEB).

The YqhD-gene was isolated from K12 E. coli and amplified by colony PCR using the YqhD (FW) and YqhD (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with DpnI for 120 minutes and subsequently purified. The YqhD amplicon was digested with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. E. coli Top 10 were 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 which were verified by sanger sequencing.