Team:TU Darmstadt/Notebook/sec1/K1602027
K1602027 - HistidinTag-Scaffold (His-Scaffold)
Last year’s iGEM Team (2014, TU Darmstadt) designed a scaffold containing three binding domains (PDZ, GBD, SH3). To the scaffold GSP sequence a Histidin-Tag was added by PCR using the oligonucleotides VR and 40 (Figure 1). The PCR product was digested with DpnI. Afterwards the sample was purified. The BioBrick prefix was added with another PCR using oligonucleotides 40 and VR (Figure 2). The PCR product was digested with DpnI and purified. The construct was digested with EcoRI and PstI and ligated into pSB1C3. Competent E.coli Top 10 cells were transformed via heat shock. A colony PCR with VF2 and VR was performed (Figure 3). Positive colonies were inoculated and the plasmids were extracted.
Figure 1 PCR of Histidin-Tag added to the Scaffold sequence. The size of the amplified product (1) was around 1.0 kbp. DNA marker: 2-Log DNA Ladder.
Figure 2 PCR of the BioBrick prefix added to the His-Scaffold sequence. The size of the amplified product (1) was around 1.1 kbp. DNA marker: 2-Log DNA Ladder.
Figure 3 Colony PCR of Prefix-His-Scaffold cloned into pSB1C3. All colonies (1-3) contained the insert of around 1.1 kbp. DNA marker: 2-Log DNA Ladder.