Tgradient thermocycler

mRNA isolation was performed with InnuPREP RNA mini kit (Analytikjena, cat. no. 845-KS-2040250, Jena, Germany) according to the supplier’s protocols. The quantity of RNA was measured on an Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany) and considered pure if the absorbance at 260 nm/280 nm was more than 1.8. The concentration of 0.4 µg RNA/10 µL of dH₂O was used. cDNA synthesis was performed using High Capacity kit (Applied Biosystems, cat. No. 4368814, Foster City, CA, USA) according to the manufacturer’s instructions on a TGradient thermocycler (Biometra, Göttingen, Germany).

The pH and temperature effects on the enzyme activity were evaluated by preparing the substrate suspensions in McIlvaine's buffer in the pH range of 2.2‐8.0 at 30 °C and in the temperature in the range of 30–65 °C at pH 3.0 for ABTS and at pH 4.0 for 2,6‐DMP and guaiacol. The T₅₀ value was assessed in a gradient thermocycler (Biometra T Gradient Thermocycler), corresponding to the temperature at which the enzyme retains 50% of its activity after a 10‐min incubation. Subsequently, 176 μL aliquots were used in triplicate assays at temperatures between 25 and 82 °C. After a 10‐min incubation, samples were immediately chilled in ice water for 10 min. Thereafter, 2 μL of each sample (the protein concentration was 5 μg·mL⁻¹) was used to measure the enzyme activity.

Equal amounts of siRNA (negative control and hnRNP H) treated U87 RNA (100–300 ng), and equal amounts of patient cerebellum polyA RNA (50 ng) were reverse transcribed using Maxima Reverse Transcriptase according to manufacturer’s instructions (Fermentas) with 4 μM random pentamer (Invitrogen). The resulting cDNAs were then diluted (1:10 for siNC/H and 1:2 for polyA cerebellum) and used in PCR with 1X standard Taq reaction buffer, 1.5 mM MgCl₂, 200 μM dNTPs, including 0.4 pmol α-³²P dCTP, (3000 Ci/mmol, Perkin Elmer), 0.8 μM each forward and reverse primer (Supplementary file 3), and 3 U/25 μl reaction recombinant Taq polymerase (Invitrogen). PCR was performed using a Biometra T Gradient thermo cycler programmed for 33 cycles of 20 s denaturation at 95°C, 30 s annealing at 55°C, and 80 s extension at 72°C, with an initial 2 min denaturation step at 95°C and a final 5 min extension at 72°C. 6X DNA load dye was added and PCR products were loaded onto a 6% native acrylamide gel, run for 2 hr at 200 V, transferred to Whatman paper, dried, and exposed to phosphorimager. Bands were quanitifed using ImageQuant, and mean percent inclusions (3–4 replicates per patient) were graphed, and statistically analyzed by t test, using Graphpad for Prism software.

Genomic DNA was isolated from mutants and TAIL-PCR [44 (link), 45 (link)] performed using AD and T-DNA end primers [39 (link), 45 (link)] by TGradient Thermocycler (Biometra, Göttingen, Germany). Following tertiary TAIL-PCR, fragments were purified, and then sequenced directly. The flanking sequences obtained were subjected to a BLAST search using the Arabidopsis Information Resource (TAIR, http//:www.arabidopsis.org). Finally, specific primers, 5’-ACAGATATGTACAAACCTCACTAG-3’ (T3K9-RB) and 5’-GGAGGAGGAGAACGGTCAAAGCGG-3’ (T3K9-LB), were designed by MacVector ver. 12 (Cary, NC, USA) and used in combination with T-DNA-specific primers, 5’-CGTTCAAGATGCCTCTACCGACAG-3’ (RB2) and 5’-TGGGATTGTGCGTCATCCCTTACG-3’ (LB2), to amplify specific fragments; these were subsequently sequenced to confirm the insertion sites.

Total RNA was isolated from feline pancreas (RNeasy Micro Kit, Qiagen) and reverse transcribed into cDNA (Omniscript Reverse Transcription Kit, Qiagen). PCR was carried out with the Expanded High Fidelity PCR System (Roche Applied Science) and a TGradient Thermocycler (Biometra) using various primers sets (Table 2) and feline pancreatic cDNA as the template. Thermal cycling parameters were 94°C for 5 min, followed by 34 cycles of amplifications at 94°C for 30 s, annealing for 30 s, 72°C for 45 s, and 72°C for 7 min as the final elongation step. The annealing temperature used varied depending on the primers used.

Uniplex PCR for PRSV, PLDMV, or PapMV was carried out in a 25 μL mixture containing 12.5 μL 2 × Eco Taq PCR SuperMix (+dye) (TransGen Biotech, Beijing, China), 0.5 μL of specific primers (20 μM) (Table 1), 2 μL cDNA template, and 10.5 or 9.5 μL sterile deionized H₂O. In a negative control reaction, nuclease free water or cDNA from healthy (virus-free) plant was used as the template. To determine the best annealing temperature for a correct amplification preventing non-specific products, a gradient PCR was performed in a TGradient Thermocycler (Biometra, Goettingen, Germany) using the following parameters: one cycle at 94 °C for 3 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 50–65 °C (with an increase of 2 °C per tested sample) for 30 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 5 min. PCR products were examined by electrophoresing 10 μL aliquots on 1.5% agarose gels in TAE buffer. Each amplified viral target fragments was cloned into the pMD18-T vector (Takara), and the resulting plasmid construct pPRSV-953, pPRSV-613, pPRSV-515, pPLDMV-355, pPapMV-476, and pPapMV-205, was confirmed by sequencing (Life Technologies, Guangzhou, China) and BLASTN at NCBI [41 ].

Genomic DNA extractions, plasmid preparations, PCR reactions, ligations, transformations, and other standard molecular biology techniques were carried out as described elsewhere (Sambrook & Russell 2012 ) or following the instructions of the supplier. PCR experiments were carried out by using T Gradient thermocycler (Biometra, Germany) and Ex Tag DNA polymerase (Takara Bio, Inc., Japan). Genomic DNA extracted from S. elodea was used as the template. PCR products were purified using QIAquick PCR purification kit (Qiagen, Germany). E. coli and S. elodea cells were transformed by electroporation using Electro Cell Manipulator (BTX Technologies Inc., USA).