Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602001"

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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 <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 BBa_B0034 ribosomal binding site was added upstream of the <em>xylC</em>-gene by cloning said gene in the <em>pSB1A2‑</em>vector containing the RBS.</p>
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<p>The <em>pSB1A2‑</em>vector was cut using <em>Spe</em>I<em>‑</em> and <em>Pst</em>I‑restriction enzymes and later dephosphorylated. The gene was digested using <em>Xba</em>I and <em>Spe</em>I and ligated in the vector with <em>T4</em>‑ligase. After transformation in <em>E. coli </em>Top10 the resulting colonies were screened for positive clones by performing colony‑PCRs with the VF2 and VR standard-oligonucleotides. The plasmids of positive clones were purified and the inserts verified by sanger-sequencing. Afterwards the <em>pSB1A2‑</em>vector was replaced by the <em>pSB1C3</em> registry standard vector using <em>EcoR</em>I and <em>Spe</em>I. The dephosphorylation and ligation was performed.</p>
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Revision as of 17:20, 17 September 2015

K1602001 - D-xylonolactonase with strong RBS (B0034-xylC)



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 BBa_B0034 ribosomal binding site was added upstream of the xylC-gene by cloning said gene in the pSB1A2‑vector containing the RBS.

The pSB1A2‑vector was cut using SpeI‑ and PstI‑restriction enzymes and later dephosphorylated. The gene was digested using XbaI and SpeI and ligated in the vector with T4‑ligase. After transformation in E. coli Top10 the resulting colonies were screened for positive clones by performing colony‑PCRs with the VF2 and VR standard-oligonucleotides. The plasmids of positive clones were purified and the inserts verified by sanger-sequencing. Afterwards the pSB1A2‑vector was replaced by the pSB1C3 registry standard vector using EcoRI and SpeI. The dephosphorylation and ligation was performed.