Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602009"

 
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<h3>K1602003 - <small>D</small>-xylose dehydratase xylB</h3>
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<h3>K1602009 - <small>D</small>-Xylose Dehydratase (xylB)</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>xylB</i>. The size of the amplified product was around 950 bp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
 
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The <i>xylB</i>-gene was synthesized by GeneArt and amplified through PCR using the <i>xylB</i> (FW) and <i>xylB</i> (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with Dpn1 for 120 minutes and subsequently purified. The <i>xylB</i> amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. <i>E.Coli</i> Top 10 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 by colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by sanger sequencing.
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<p>The <em>xylB</em>-gene was synthesized by GeneArt and amplified by PCR using the <em>xylB</em> (FW) and <em>xylB</em> (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with <em>Dpn</em>I for 120 minutes and subsequently purified. The <em>xylB</em> amplicon was cut with <em>EcoR</em>I and <em>Pst</em>I and ligated into a pSB1C3 vector using T4-ligase. <em>E. coli</em> Top 10 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 by colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by sanger sequencing.</p>
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Latest revision as of 19:39, 18 September 2015

K1602009 - D-Xylose Dehydratase (xylB)


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

Figure 1 PCR of xylB. The size of the amplified product was around 950 bp. DNA marker: 2-Log DNA Ladder (NEB).

The xylB-gene was synthesized by GeneArt and amplified by PCR using the xylB (FW) and xylB (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with DpnI for 120 minutes and subsequently purified. The xylB amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. E. coli Top 10 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 by colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by sanger sequencing.