Team:TU Darmstadt/Notebook/sec1/K1602008

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K1602008 - Aldose Reductase with strong RBS (B0034-GRE3)

Figure 1 PCR of RBS-GRE3 (X). The size of the amplified product was around 1.0 kbp. DNA marker: 2-Log DNA Ladder (NEB).

The GRE3-gene was synthesized by GeneArt and amplified by PCR using the GRE3-RBS (FW) and GRE3 (REV) oligonucleotides to amplify it including the B0034 ribosomal binding site. The PCR product was verified by agarose gel electrophoresis and subsequently purified. The GRE3 amplicon was cut with EcoRI and PstI and ligated into a pSB1C3 vector using T4-ligase. The ligation product was transformed into E.coli Top 10 by heat shock transformation and the cells were spread out on a CMP agar plate. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Plasmid DNA was isolated from positive clones which were verified by Sanger sequencing.