Phusion high fidelity pcr kit

The p35S::GFP-IgBBX constructs were introduced into tobacco epidermal cells via the Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaf method [26 (link)]. The plasmid of IgBBX for transient transformation was generated using the Invitrogen Gateway system, according to the manufacturer’s instructions. The IgBBX ORFs, lacking the stop codon, were amplified using a Phusion^®High-Fidelity PCR kit (New England Biolabs, Ipswich, MA, USA) (primers listed in Table S1), then inserted into the pMD19-T easy vector (TaKaRa, Tokyo, Japan) to allow its sequencing-based validation. Each pENTR^TM1A-IgBBX plasmid was previously subjected to the LR Clonase™ II enzyme mix (Invitrogen, Carlsbad, CA, USA) reaction to obtain GFP-fused constructs using the binary vector pMDC43, resulting in the plasmid p35S::GFP-IgBBX. For transient expression, Agrobacterium tumefaciens strain EHA105 carrying the pMDC43 and pMDC43-IgBBX was grown separately to OD600 = 0.8 and coinfiltrated with the p19 strain into leaves of five-week-old N. benthamiana. The YFP fluorescent signals were monitored 48 to 72 h after infiltration using a confocal laser scanning microscopy (LSM 700, Carl Zeiss).

S. cerevisiae strains used in this study are listed in Supplementary Table S6, primers are listed in Supplementary Table S7, and plasmids in Supplementary Table S8. To create the Sdo1^ts strain, the conditional TS18 intein^{28 (link),40 (link)} was amplified by PCR from plasmid pS5DH-G4MINT (gift from N. Perrimon) and inserted between the SDO1 codons for K73 and C74 by homologous recombination. For the generation of Tif6-GFP mutants, site-directed mutagenesis of the pTIF6-GFP plasmid was performed using the Phusion High-Fidelity PCR kit (NEB) and transformed into XL1-Blue Electroporation-Competent cells (Agilent).

Total RNA was isolated from 100 mg of barley anthers using Trizol (Invitrogen). One µg of RNA was employed for cDNA synthesis using the Invitrogen FirstStrand cDNA Synthesis Using SuperScript II RT kit following manufacturer’s instructions. cDNA samples were used for PCR amplification using the NEB Phusion High Fidelity PCR Kit and Sanger resequencing of the coding sequence of barley MTOPVIB. For primer sequences see Online resource 1.

Top eight samples were taken to perform single-molecule real-time sequencing (SMRT). The cDNAs were synthesized from viral RNAs by reverse transcription using Uni12 and Uni13 primers (Bi et al., 2016 (link)). The PCR was performed using a Phusion High-Fidelity PCR Kit (New England Biolabs) utilizing the barcoded influenza A virus general primers (Supplementary Table S1) (Mei et al., 2016 (link)). The concentration of PCR product was quantified by the Agilent Technologies 2,100 bioanalyzer. The two corresponding volumes of PCR products (containing equal mass of dsDNA) were mixed into one sample and quantified in the bioanalyzer again. About 2–3 μg mixed sample was used to SMRTbell library construction following the 2 kb template preparation protocol (Roberts et al., 2013b (link)). The sequencing was performed on a PacBio RS II instrument (Pacific Biosciences, USA) with one SMRT Cell used for each library, using P6/C4 chemistry with a 4 h movie (Bull et al., 2016 (link)). SMRTbell adapter sequences were removed and circular consensus sequence (CCS) reads were achieved with SMRT Analysis v2.3 (Roberts et al., 2013a ).

RNA was extracted using standard Trizol RNA extraction. cDNA was synthesized from 500 ng total RNA in a 10 μl reaction, using Superscript VILO cDNA synthesis kit (Invitrogen) following manufacturer’s instructions. Splicing profiles were monitored by PCR using primers in flanking exons. For each PCR, 1 μl diluted cDNA (1/8) was used as template in a 10 μl PCR reaction using Phusion High-Fidelity PCR Kit (NEB, UK) following manufacturer’s instructions. Splicing profiles were monitored and quantified using the Qiaxcel capillary electrophoresis system (Qiagen) and PSI was calculated as described previously18 (link). All primers used for splicing assays are provided in Supplementary Data 3.