Genejet plasmid maxiprep kit

The pHR’ lentiviral transfer plasmid was used as a template to produce a lentiviral transfer plasmid that encodes IL-15/IL-15Rα. The construct contains a murine Igκ-signal peptide, the IL-15 sequence (NCBI Reference Sequence: NP_000576.1) and was connected via a P2A self-cleaving site to a Igκ-signal peptide followed by the IL-15Rα sequence (NCBI Reference Sequence: NP_002180.1) and a hemagglutinin (HA)-tag.
The gene fragments were designed in silico using SnapGene (Dotmatics, USA) and subsequently codon optimized using the IDT codon optimization tool. We added 3’ and 5’ sequences that were homologous to the pHR’ plasmid insertion site and ordered the synthetic gene fragments (gBlocks, Integrated DNA Technologies, USA). The transfer plasmid was opened with restriction enzymes EcoRI and BamHI (Thermo Fisher Scientific, USA) and the gBlocks were ligated using the NEBuilder HiFi DNA Assembly mix (New England Biolabs, USA). Competent bacteria (New England Biolabs) were transformed, expanded and plasmid DNA was isolated using the GeneJET Plasmid Maxiprep Kit (Thermo Fisher Scientific). Quantities of plasmid DNA were evaluated using a spectrophotometer.
The lentiviral transfer plasmids encoding K2-Fc or R3-Fc^{21 (link)}, an antibody-variant of the nanobody that binds the idiotype of 5T2 myeloma cells (R3B23)^{22 (link)}, were previously described.

The sequence of pri-miR-181c-5p was amplified from the genomic DNA of human Peripheral Blood Mononuclear Cells (PBMCs), extracted by QIAamp genomic DNA kit (QIAGEN, USA), using Thermo Scientific DreamTaq Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA) and forward primer: 5′-TCGA-GGATCC- ACTTAAGGAGCGGGCTTGAG-3′ and reverse primer: 5′-TCGA-GCTAGC- TCACAACCCACCGACAACA-3′, according to the manufacturer’s instruction. The PCR for pri-miR-181c-5p amplification was carried out as follows: 95 °C for 5 min; and 35 cycles of 94 °C for 15 s, 54 °C for 30 s, and 72 °C for 30 s. The amplified product was subsequently cloned in a pEGP-miR vector to form pmiR-181c-5p with the aid of Thermo Scientific T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA). The cloned constructs were transformed subsequently into the cells of Escherichia coli TOP10 and confirmed by sequencing. Following the manufacturer’s guidelines, the verified clone was purified by an endotoxin-free GeneJET Plasmid Maxiprep Kit from Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA). The Thermo Fisher Scientific Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the concentration and purity of the pmiR-181c-5p construct. The pEGP-miR null vector (pNull) was considered a control vector.