Team:TU Darmstadt/Notebook/sec1/K1602003

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K1602003 - Cis-Aconitate Decarboxylase (cadA)

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 cadA-gene was synthesized by GeneArt and amplified by PCR using the cadA (FW) and cadA (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis and subsequently purified. The cadA amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. E.coli Top 10 were transformed with the ligation product via heat shock. The cells were spread out on a CMP agar plate. Positive clones were determind by colony PCR using VF2 and VR oligonucleotides. After plasmid purification they were verified by Sanger sequencing.