Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602000"
| Line 9: | Line 9: | ||
</figure> | </figure> | ||
| − | <p align="justify" | + | <p align="justify"> |
| − | The BBa_B0034 ribosomal binding site was added upstream of the <em>xylB</em>-gene by cloning | + | The BBa_B0034 ribosomal binding site was added upstream of the <em>xylB</em>-gene by cloning the gene into the <em>pSB1A2‑</em>vector containing the RBS. |
| − | + | 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 construct was digested using <em>Xba</em>I and <em>Spe</em>I and ligated into the vector with <em>T4</em>‑ligase. After transformation in <em>E. coli </em>Top 10 the resulting colonies were screened for positive clones by performing colony PCRs with the VF2 and VR 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 <em>EcoR</em>I and <em>Spe</em>I. The dephosphorylation and ligation was performed as described before.</p> | |
</div> | </div> | ||
</html> | </html> | ||
Revision as of 17:23, 17 September 2015
K1602000 - D-xylose dehydratase with strong RBS (B0034-xylB)
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 xylB-gene by cloning the gene into the pSB1A2‑vector containing the RBS. The pSB1A2‑vector was cut using SpeI‑ and PstI‑restriction enzymes and later dephosphorylated. The construct was digested using XbaI and SpeI and ligated into the vector with T4‑ligase. After transformation in E. coli Top 10 the resulting colonies were screened for positive clones by performing colony PCRs with the VF2 and VR 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 as described before.