Team:TU Darmstadt/Notebook/sec1/K1602021
K1602021 - HistidinTag-Green fluorescent protein (His-GFP)
Competent E. coli Top 10 cells were retransformed via heat shock with pSB1C3 harbouring GFP provided by the iGEM Registry (BBa_E0040). The plasmids were extracted and used as templates for a PCR with oligonucleotides 42 and VR. The PCR product was verified by gel electrophoresis (Figure 1) and digested with DpnI. The purified product was digested with EcoRI and PstI and ligated into pSB1C3. Competent E. coli Top 10 cells were transformed via heat shock and colonies were screened by colony PCR with oligonucleotides VF2 and VR (Figure 2).
Figure 1 Standard PCR of Prefix-His-Tag-GFP (3). The size of the amplified product was around 0.9 kbp. DNA marker: 2-Log DNA Ladder (NEB).
Figure 2 : Colony PCR of Prefix-His-Tag-GFP (1+2). Colonies 1 and 2 contained the insert of around 0.9 kbp. DNA marker: 2-Log DNA Ladder (NEB).