Team:TU Darmstadt/Notebook/sec1/K1602004

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K1602004 - Aldose Reductase (GRE3)

Figure 1 PCR of 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 (FW) and GRE3 (REV) oligonucleotides. The PCR product was verified by agarose gel electrophoresis and subsequently purified out of the gel because of unknown amplificates in the range of a few hundred kbp. The purified GRE3 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. Clones were screened with colony PCR using VF2 and VR oligonucleotides. Positive clones were determind by colony PCR using VF2 and VR oligonucleotides. After plasmid purification they were verified by Sanger sequencing.