Innuprep plasmid mini kit

Genes encoding SOPP, CreiLOV and iLOV harboring an NdeI and XhoI restriction site at the respective 5′- and 3′- end were obtained by commercial gene synthesis (Eurofins Genomics, Ebersberg, Germany). Subsequently, the synthetic DNA fragments were cloned into the NdeI and XhoI sites of the pET28a vector (Novagen, distributed by Merck KGaA, Darmstadt, Germany). DsFbFP M49I was generated by overlap extension PCR using the pET28a DsFbFP vector DNA^{65 (link)} as template and the oligonucleotide primers DsFbFP-up: 5′-CAGCCATATGCGCAGAC-3′, DsFbFP-dn: 5′- GTGCTCGAGTCAGACCGGG-3′, DsFbFP 49I-up: 5′-CAACCCGATTATCTATGTC-3′ and DsFbFP 49I-dn: 5′-GACATAGATAATCGGGTTG-3′. The resulting final PCR fragments were hydrolyzed with NdeI and XhoI and ligated into the respective sites of the pET28a vector. DNA cloning was conducted using the Escherichia coli strain DH5α^{89 (link)} and DNA plasmid isolations from bacterial cells were performed using the commercial innuPREP Plasmid Mini Kit (Analytik Jena, Jena, Germany), as described by the manufacturer. All final vector constructs were verified by DNA-sequencing (Eurofins Genomics, Ebersberg, Germany).

All chemicals were acquired from Carl Roth GmbH & Co. KG (Karlsruhe, Germany) if not mentioned otherwise. Standard molecular methods were conducted as described before by Sambrook et al. [28 ]. Chromosomal DNA and plasmid DNA were purified by application of innuPREP Bacteria DNA Kit and innuPREP Plasmid Mini Kit, respectively (Analytik Jena AG, Jena, Germany). All primers used for PCR reactions were synthesized by Eurofins Genomics (Ebersberg, Germany). DNA fragments were amplified by polymerase chain reactions using Phusion High-Fidelity DNA Polymerase (New England BioLabs, Frankfurt am Main, Germany). PCR reactions were performed with thermo cycler (prqSTAR 96X VWR GmbH, Darmstadt, Germany). Amplified PCR products were extracted with QIAquick PCR & Gel Cleanup Kit (QIAGEN GmbH, Hilden, Germany).