Topo xl 2 pcr cloning kit

Real-time qPCR for DCH was performed as described previously [7 (link)]. For absolute DCH DNA quantification, a 1.4 kb-long fragment of the polymerase region of the Australian reference strain AUS/2016/Sydney was cloned using the TOPO XL-2 PCR cloning kit (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). Tenfold dilutions at 10⁰–10⁹ copies of plasmid per reaction were used to generate the standard curve. Standards and samples were run in triplicate. Ultrapure water and salmon sperm DNA were used as negative controls. Reactions were 25 μL, comprising 1 μL of neat template DNA plus 9 μL of water; or 10 μL plasmid standard and 15 μL of master mix, comprising IQ Supermix (Bio-Rad Laboratories SRL, Segrate, Italy), containing 0.6 μmol/L of each primer and 0.1 μmol/L of probe. Thermal cycling consisted of activation of Taq DNA polymerase at 95 °C for 3 min and 42 cycles of denaturation at 95 °C for 10 s and annealing-extension at 60 °C for 30 s. A sample was defined as positive if ≥10 copies of DCH DNA were detected in at least ≥ two of three replicates with a Ct value of ≥38.5. Thus, our PCR assay had a lower limit of quantification of 2500 copies/mL of blood. The cut-off for R-squared was 0.980 and for efficiency was 90–110%.

For SNP variant identification, the TOPO XL-2 PCR Cloning Kit (Invitrogen) was used. Briefly, RNA isolated from SCA3 and HD cell lines (iPSCs) was reversely transcribed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) with random hexamer primers (for ATXN3) or SuperScript IV Reverse Transcriptase (Thermo Fisher) with a gene-specific primer (HTT_RT) (for HTT). Then, cDNA served as a template in PCR reactions using Platinum SuperFi polymerase included in a cloning kit. In case of HTT, PCRs were performed using PrimeSTAR GXL DNA Polymerase (Takara Bio). Primers in those PCR reactions were designed in a way to amplify whole CDSs, with fragments of both 5′ and 3′UTRs (ATXN3_Fwd and ATXN3_Rev; HTT_Fwd and HTT_Rev, sequences given in Additional File 10: Table S2). PCR products were then cloned into pCR-XL-2-TOPO vector. Generated vectors were transformed into bacterial cells provided with the kit. Transformants were analyzed through colony PCR and restriction enzyme digestion to select clones with either WT or MUT versions of analyzed genes. Positive clones were then sequenced, and SNPs allowing for discrimination between WT and MUT alleles, after additional confirmation on genomic DNA (gDNA), were identified. All identified SNPs are listed in Figs. 1b and 3b.