Ap pcr 250

For all Pepper-based sensor RNAs, double-stranded DNA (dsDNA) templates were designed to contain a 5′ T7 promoter to be used for in vitro transcription. dsDNA templates were prepared from single-stranded DNA oligos (Tsingke Biotechnology). DNA templates were amplified by polymerase chain reaction (PCR) using Taq DNA polymerase (Vazyme P112) and checked for quality using 1.5% agarose gel electrophoresis. PCRs were purified with the PCR purification kit (Axygen AP-PCR-250).
In vitro transcription reactions using the T7 High Yield RNA Transcription Kit (Vazyme DD4201) were carried out according to the manufacturer's instruction. Transcription reactions were terminated by treatment with RNase-free DNase I at 37°C for at least 15 min. An 80 μl aliquot of RNase-free water was added followed by an equal volume of water-saturated phenol–chloroform, and this was then centrifuged at 4°C, 12 000 rpm for 15 min. The supernatant liquid was aspirated with sodium acetate and 2 times the volume of anhydrous ethanol, and left to settle at –20°C for 30 min. Afterwards, the precipitate was washed with 75% anhydrous ethanol prepared with enzyme-free water and centrifuged for 5 min, and then dissolved in enzyme-free water.

The SMN2 mini-gene was constructed using pCI-SMN1, pCI-SMN2, pEGFP-SMN1, and pEGFP-SMN2 according to a previous method described by the Hua lab (Hua et al., 2018). The pCGT7 plasmid was used to overexpress NOVA1. First, NOVA1 was amplified using the following primers: hNOVA1 Nhe1-forward: 5′-ATG CTA GCA TGA TGG CGG CAG CTC CC-3′, hNOVA1 Kpn1-reverse: 5′-ACG GTA CCT CAA CCC ACT TTC TGA GG-3′. The polymerase chain reaction (PCR) mixture included 4 μL 5× Phusin high fidelity buffer, 0.4 μL 10 mM deoxy-ribonucleoside triphosphates, 1.2 μL forward primer, 1.2 μL reverse primer, 1–10 ng cDNA, 1.2 μL dimethyl sulfoxide, 0.3 μL Phusion DNA polymerase (New England Biolabs, NEB, Ipswich, MA, USA), and ddH2O to 20 μL. The reaction procedure was 35 cycles, including 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. The PCR product was purified with a PCR clean-up kit (Axygen, Union, CA, USA, Cat# AP-PCR-250) and then digested with NheI and XbaI and ligated into the XbaI and KpnI sites in pCGT7 (Additional Figure 1). The final NOVA1 overexpression plasmid was obtained through transformation, amplification, plasmid extraction, and sequencing to verify the amplification results.