Hifi hotstart dna polymerase

The RCA DNA (20 µl aliquot from each sample) was sequenced on an Illumina 4000 (Illumina) sequencer at Macrogen (Korea). The paired-end reads were de novo assembled using ABySS v2.02 [29 (link)], and contigs >250 nucleotides were analysed using blastx [30 (link)] against a local viral database. A contig of 372 nucleotides was identified in the liver sample with similarities to VP2 and VP1 of fish PyVs. Based on the sequence of this contig, a pair of abutting primers (F: 5′-CGCTGCTAAAGGAAATAAAATCAAGATATGGGAAT-3′; R: 5′-ACTGGAATTTTAGGCAAATCTATAGCTGACGTTAG-3′) were designed to recover the full genome of this putative PyV. A 0.5 µl sample of the RCA reaction was used as a template for PCR amplification using the abutting primers and Kapa HiFi Hotstart DNA polymerase with the following thermal cycling protocol: 95 °C for 3 min; 25 cycles of 98 °C for 20 s, 60 °C for 15 s, 72 °C for 5 min, and a final extension of 72 °C for 6 min. The amplicon was resolved on a 0.7 % agarose gel, the ~5 kb product was excised, gel-purified and cloned into the pJET1.2 plasmid vector (ThermoFisher, USA). The recombinant plasmid was Sanger-sequenced by primer walking at Macrogen (Korea), and the Sanger sequence contigs were assembled using DNA Baser V4 (Heracle BioSoft S.R.L., Romania).

Mouse monoclonal antibodies against AQP1 (ab9566) and RIPK1 (ab72139) were purchased from Abcam (Cambridge, MA). Mouse monoclonal antibodies against RIPK1 (610458), caspase-8 (9746), and RIPK3 (sc-374639) were bought from BD Biosciences (San Jose, CA), Cell Signaling Technology (Danvers, MA), and Santa Cruz Biotechnology (Dallas, TX), respectively. The rabbit monoclonal antibody against p-MLKL (S345) (ab196436), p-RIPK3 (S227) (ab209384) and p-RIPK3 (S232) (ab195117), and rabbit polyclonal antibody against RIPK1 (ab106393) and MLKL (ab194699) were purchased from Abcam. The rabbit polyclonal antibody against cleaved caspase-3 (9661) and caspase-3 (9662) were bought from Cell Signaling Technology. The rabbit monoclonal anti-β-actin antibody (AC026) and anti-α-tubulin antibody (AC013) were from Abclonal (Wuhan, China). The cell transfection regent (F231-02) was purchased from TransGen Biotech (Beijing, China). The point mutation plasmids of RIPK1 were constructed using the HiFi HotStart DNA Polymerase (KAPA Biosystems, Roche, Switzerland).

Construction of DNA plasmids to generate in vitro transcription (IVT)-produced mRNA was performed as previously described^{62 (link)}. Briefly, DNA constructs containing genes of interest were ordered from Dharmacon or Addgene. Primers were designed that spanned the open reading frame (ORF) of the genes of interest. PCR reactions were performed using HiFi HotStart DNA polymerase (KAPA Biosystems) per the manufacturer’s instructions. Amplified ORFs were treated using T4 polynucleotide kinase (New England Biolabs). PCR products were then purified using QIAquick spin columns (Qiagen). Blunt ligations between the PCR products and a template plasmid (pORF-in) that incorporates generic 5’ and 3’ untranslated regions (UTRs) and a poly-A tail, as previously described^{62 (link)}, were performed using T4 ligase (New England Biolabs). Newly ligated plasmids were propagated using the TOPO TA cloning kit (ThermoFisher Scientific) and verified by Sanger sequencing in the KI gene facility (Stockholm, Sweden).

Forward primer 5′-TACCTGATGGTTCTTTAAAATTGGG (designed for this study) and reverse primer 5′-CATAAGCTAGACCTTGAAGGATGTC38 (link) were used for isolation of the full-length coding region of Pi54 alleles as annotated for Pi54 allele from cultivar Tetep37 (link). PCR was performed with an initial denaturation at 95 °C for 5 minutes; followed by additional denaturation at 98 °C for 20 seconds, annealing at 61 °C for 20 seconds, extension at 72 °C for 1 minute (these three steps repeated for 35 cycles); followed by final extension at 72 °C for 2 minutes, using KAPA HiFi HotStart DNA polymerase (high fidelity proof-reading enzyme). The amplified products were cloned using pJET1.2 blunt end cloning vector.

Constructs for promoter activity analysis of CV1 and CV2 were generated by cloning the1500 bp promoter sequences upstream of the start codon of each gene into pBGWFS7 vector (Karimi et al., 2002 (link)), to regulate GUS expression. Promoter sequences were PCR amplified from genomic DNA of soybean TJS2049, using Kapa HiFi Hotstart DNA polymerase. Fragments of approximately 3000 bp were amplified using primers Fw 5’-CATAGATACTCATCGAAAATGG-3’ and Rv 5’-GTTGAGGATGTTTTGGGGGAA-3’ for Pro-CV1, and Fw 5’-GCCAAGTCCCAAAAGTGAACA-3’ and Rv 5’-GTGGCTTTGCGGGGTTGAAAGAAGCGT-3’ for Pro-GmCV2. BamHI and XhoI restriction sites were added to 1500 bp fragment through nested PCR using primers Fw 5’-AATGGATCCTTATAGATGATAAAACAG-3’ and Rv 5’-TGGTCTCGAGGGTGAGAGTTGAGTTGAG-3’ for Pro-CV1 or Fw 5’-AATGGATCCTTATTTTGGTCAGGGATT-3’ and Rv 5’-TGGTCTCGAGGGTGAGAGTTGAGACAAT-3’ for Pro-GmCV2. PCR fragments were cloned into BamHI and XhoI restriction sites in pENTR-2B (Invitrogen-Thermo Scientific) and sequenced. Clones were subsequently recombined into pBGWFS7, using Gateway LR Clonase II (Invitrogen-Thermo Scientific). The constructs were introduced into Agrobacterium tumefaciens, strain C58C1 (Deblaere et al., 1985 (link)) by electroporation and used for Arabidopsis transformation.

A fragment of the human IRF6 gene, comprising exon 7 (of nine) and 309 bp of intron 6 and 428 bp of intron 7, was amplified by polymerase chain reaction (PCR) using HiFi HotStart DNA Polymerase (Kapa Biosystems) and human genomic DNA isolated from an unaffected control individual with the following primer pair containing restriction site linkers: hIRF6-I6 forward (5′-CAATCTCGAGCCGACTCAGTCAG TATAGCGTGGG-3′; XhoI site underlined) and hIRF6-I7 reverse (5′-AGCTCTAGATGTAACTCCCTTC TTTGTTGCC-3′; XbaI site underlined). The purified product was double digested with XhoI and XbaI and cloned into a similarly digested pET01 exon trap vector (MoBiTec, Goettingen, Germany) to generate the wildtype (WT; c.921C) construct.
Plasmids for the expression of Splice Regulatory Factor 1 (pcDNA-FLAG-SF2; Addgene 99021) and Splice Regulatory Factor 2 (pcDNA3.1-SC35-cMyc; Addgene 44721) were separately co-transfected with the empty pET01 vector, or either the WT (c.921C) or mutant (c.921T) vectors into COS7 cells and total RNA was extracted two days post transfection (n = 6 per group), as described above. Following cDNA synthesis, PCR products were amplified as previously described, separated on a 1.2% agarose gel and quantified as above.