Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602010"
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| − | <h3> | + | <h3>K1602010 - D-Xylonolactonase (xylC)</h3> |
<html><div class="contentSection"> | <html><div class="contentSection"> | ||
<figure class="rightFig" style="width:30%"> | <figure class="rightFig" style="width:30%"> | ||
| − | <img | + | <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"> |
| − | <figcaption><br><b>Figure 1</b> PCR of | + | <figcaption><br><b>Figure 1</b> PCR of <i>xylC</i>. The size of the amplified product was 1 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption> |
</figure> | </figure> | ||
| + | <p align="justify"> | ||
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The <I>xylC</I>-gene was synthesized by GeneArt and amplified | The <I>xylC</I>-gene was synthesized by GeneArt and amplified | ||
| − | + | by PCR using the <I>xylC</I> (FW) and <I>xylC</I> (REV) | |
oligonucleotides. The PCR product was verified by agarose gel | oligonucleotides. The PCR product was verified by agarose gel | ||
| − | electrophoresis, digested with | + | electrophoresis, digested with <i>Dpn</i>I for 120 minutes and subsequently |
| − | purified. The <I>xylC</I> amplicon was cut with | + | purified. The <I>xylC</I> amplicon was cut 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 | + | ligated into a pSB1C3 vector using T4-ligase. <I>E. coli</I> Top 10 |
has been transformed with the ligation product by heat shock | has been transformed with the ligation product by heat shock | ||
transformation and the cells were spread out on a CMP agar plate. | transformation and the cells were spread out on a CMP agar plate. | ||
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Plasmid DNA was isolated from positive clones which were verified by | Plasmid DNA was isolated from positive clones which were verified by | ||
sanger sequencing. | sanger sequencing. | ||
| + | </p> | ||
</div></html> | </div></html> | ||
Latest revision as of 19:40, 18 September 2015
K1602010 - D-Xylonolactonase (xylC)
Figure 1 PCR of xylC. The size of the amplified product was 1 kbp. DNA marker: 2-Log DNA Ladder (NEB).
The xylC-gene was synthesized by GeneArt and amplified by PCR using the xylC (FW) and xylC (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis, digested with DpnI for 120 minutes and subsequently purified. The xylC 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.