Faststart essential dna green master reagent

Cells from the ARTP-ALE derived strain FJNU-A26 and the initial strain FJNU-6 were collected at 30 h, 42 h, 76 h, 105 h, and 140 h from 5-L fermenters under constant pH 7.0 conditions to extract their total RNA using an RNA plus Kit (Takara Biotechnology, Dalian, China) and then reverse transcribed into cDNAs using a ProtoScript II First-Strand cDNA Synthesis Kit (Invitrogen, Massachusetts, USA). The qRT-PCR assay was conducted using a three-step protocol with the LightCycler® 96 system (Roche, Maryland, USA) and FastStart Essential DNA Green Master reagent (Roche, Maryland, USA), and the primers used in this assay are listed in Additional file 1: Table S1. Two genes, gapdH and recA, were used as reference genes, and the target genes transcribed from parallel cDNA samples were assayed 3 times. Finally, gene expression was calculated using LightCycler^® Software (version 1.1.0.1320).

HeLa cells, cultured in DMEM (10% FBS 1% Pen/Strep), were plated in 6‐well plates. 24 h later, cells were counted and transduced with vector particles at a particle per cell ratio of 1,000. 24 h later, cells were harvested by extensive trypsin treatment and PBS washing steps to remove any membrane bound vector particles. Cells were counted and 3–5 × 10⁵ cells were used for subcellular fractionation, while 10⁵ cells were analysed by flow cytometry (CytoFlex S platform, Beckman Coulter) to determine transduction efficiency. Subcellular fractionation was performed as previously described (Rossi et al, 2019). Membrane, cytosolic and nuclear fractions were collected. Purity of fractions was confirmed by Western blot using anti‐Rab 5 (1:100, sc46692, Santa Cruz), anti‐Tubulin (1:5,000, T5198, Sigma Aldrich), anti‐Lamin B1 (1:5,000, 16048, Abcam) and anti‐Calreticulin (1:100, PA3‐900, Affinity BioReagents) antibodies. Fractions were subjected to DNA isolation (Qiagen, DNeasy Tissue kit) followed by qPCR analysis (FastStart essential DNA green master reagent, Roche) using CMV promoter‐specific primers on the LightCycler 96 real‐time PCR system (Roche). The specificity of target DNA amplification was confirmed by melting‐curve analysis. All samples were run in technical duplicates.