Mc easy minicircle dna production kit

Minicircles were produced using the MC-Easy™ Minicircle DNA Production Kit (System Biosciences) according to the manufacturer’s protocol. Briefly, pMC-GFP, pMC-puroR, pMC-p53-puroR, and pMC-SB100 were grown in E. coli bacterial strain ZYCY10P3S2T harboring an arabinose-inducible system for simultaneous expression of PhiC31 integrase and Sce-I endonuclease. After incubation with induction medium, intramolecular (cis-) recombination generated MC from the parental plasmid mediated by PhiC31 integrase. The remaining parental plasmid-DNA backbone was degraded by Sce-I endonuclease. MC-GFP (3.7 kb), MC-puroR (2.6 kb), MC-p53-puroR (3.7 kb), and MC-SB100 (4.5 kb) were purified from the medium using Plasmid Plus Maxi Kit (Qiagen) according to the manufacturer’s protocol.

Minicircles were produced using the MC-Easy™ Minicircle DNA production kit (System Biosciences) following the manufacturer’s instructions. HEK293-T cells were transfected with 4 μg of plasmid vectors using Lipofectamine 2000™ (Life Technologies, Waltham, MA, USA) as per the manufacturer’s instructions and incubated for 24 h at 37 °C with 5% CO₂ prior to cell harvest.
To monitor expression over time, porcine kidney IBRS2 cells were transfected, as described above, and incubated at 37 °C with 5% CO₂ for up to 72 h post-transfection. To quantify gene expression every 24 h, the growth media (1× MEM, 10% fetal bovine serum, 5% 100× Antibiotic-Antimycotic) was removed completely; cells were rinsed with 1× dPBS and replenished with fresh growth media.

The pGL3Luc2 vector was created by excision of luc2 gene with NcoI and XbaI from pGL4.10 and subcloned into a pGL3-basic vector (Promega, Leiden, The Netherlands). The mammalian expression vector pTurboFP635-N encoding far-red fluorescent protein TurboFP635 (Evrogen, Moscow, Russia) was used for the fusion of Luc2 reporter gene at the N-terminal region of the TurboFP635. Briefly, Luc2 gene was amplified from pGL4.10 vector using the sense primer 5′-CCGCTAGCAATGGAAGATGCCAAAAACAT-3′ with a NheI restriction site and antisense primer 3′-CGTGACTGCTTGCCGCCCTTCTTGGCCTT-5′ with a SalI restriction site. Subsequently, the fragment was cloned in the multiple cloning site of the pTurboFP635-N, allowing the generation of the fusion protein. The new vector was named TurboLuc. Then, the fusion protein TurboLuc was amplified using the sense primer 5′CTCTAGAGCAATGGAAGATGCCAAAAACAT-3′ with an XbaI restriction site and antisense primer 3′-TGAATTCCATCAGCTGTGCCCCAGTTTGCTA-5′ with an EcoRI site. The fragment was subcloned into pMN502A-1, a parental minicircle vector purchased from SBI (System Bioscience, Mountain View, CA, USA), and the resulting vector was called pMNTurboLuc. The minicircle vector, called MINI-pMNTurboLuc, was then purified and isolated following the protocol of the MC-easy minicircle DNA production kit (System Bioscience).