Kod fx neo polymerase

Genomic DNA was extracted from fungal hyphae grown in aseptic culture. Hyphae were frozen in liquid nitrogen, ground in a mortar, and the powder was suspended in 300 μL lysis buffer (100 mM Tris–Hc1, pH 7.5, SDS 0.5% w/v, 30 mM EDTA) and boiled for 7 min. Subsequently, 150 μL of 3 M sodium acetate was added, followed by incubation at -30°C in a freezer for 7 min. After centrifugation at 13,000 ×g for 5 min, the supernatant was extracted with an equal volume of phenol:chloroform:isoamyl alcohol (1:1:1, v/v/v) and washed with chloroform, 2-propanol, 70% (v/v) ethanol and then desiccated. Finally, the purified DNA was suspended in TE (10:1) buffer. The rDNA internal transcribed spacer (ITS) region, including ITS1 and ITS2, was amplified by PCR for sequencing using KOD FX Neo polymerase (TOYOBO, Osaka, Japan) in accordance with the manufacturer’s protocol. The primers NSI1 (5^′-GATTGAATGGCTTAGTGAG-3^′) and NLB4 (5^′-GGATTCTCACCCTCTATGA-3^′) (Martin and Rygiewicz, 2005 (link)) were used. PCR products were analyzed by agarose gel electrophoresis, extracted from the gel using the QIAEX^® II Gel Extraction Kit (QIAGEN, Velo, Netherlands) and sequenced by SigmaGenosys (Sigma-Aldrich Japan, Tokyo, Japan).

Isolated colonies were identified based on the partial sequences (c.a. 600 bp) of their 16S rRNA genes. During isolation, each of the colonies was picked with a sterile toothpick and then suspended in 35 μl TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)] in each well of a 96 well titer plate. The cells were subjected to heat shock treatment (98°C for 3 min) in order to extract the genomic DNA, which was used as template for PCR amplification. The PCR amplification of bacterial 16S rRNA gene was performed with the use of a KOD Fx Neo polymerase (TOYOBO, Tokyo, Japan), a universal primer set, 16SA1 [5′-AGAGTTTGATCMTGGCTCAG-3′] and 16SB1 [5′-TACGGYTACCTTGTTACGACTT-3′] (Fukatsu and Nikoh, 1998 (link)), and the template DNA extracted as described above. The temperature program for PCR is as follows: 94°C for 2 min, followed by 20 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 90 s. The resulting 1.5 kb amplicons were used as templates of sequencing reaction conducted with a universal primer 357F [5′-CCTACGGGAGGCAGCAG-3′] (Muyzer et al., 1993 (link)). Taxonomic assignment of the resulting sequences was double checked by the RDP multiclassifier ver. 1.1 (Wang et al., 2007 (link)) and BLASTN-search (http://ncbi.nlm.nih.gov/blast/) against GreenGene database (DeSantis et al., 2006 (link)).

The dsRNAs extracted from M. oryzae strains from the Kyushu region, NHH12P-1, MZ4-12-2, and MZ13-12-1, were used as templates for cDNA synthesis using the PrimeScript™ first-strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan) with MoV2-specific primers (Supplementary Table S1). Three overlapping PCR products were amplified using the KOD FX Neo polymerase (Toyobo, Osaka, Japan) with the primer sets Hind-T7-MoV2F/MoV2-2697R, MoV2-2657F/MoV2-detect R 4052–4073, and MoV2-detect F 3703–3725/Bam-MoV2R-re (Supplementary Table S1). The amplified PCR products were treated with 10× A-attachment mix (Toyobo, Osaka, Japan) and ligated into the pGEM T-Easy vector (Promega, Madison, United States), which was then used to transformation Hit-DH5α cells (RBC Bioscience, Taipei, Taiwan). Plasmid DNA was purified using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, United States). Sequencing was conducted using both the universal primers (M13-M3 and M13-RV) and designed sequencing primers (Supplementary Table S1). The 5′- and 3′-terminal sequences of each dsRNA segment were determined using the SMARTer® RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, United States).

USAG-1^−/− mice with a 106-bp deletion in exon 1 were produced using the CRISPR-Cas system with a C57BL/6J genetic background (fig. S1) (Macrogen Co. Ltd., Seoul, South Korea). Dental anomalies similar to those described in previous reports (10 ), including incisal supernumerary teeth, fused maxillary molars, and supernumerary mandibular molars, were observed in USAG-1^−/− mice. EDA1-deficient mice (Tabby6: C57BL/6J A^w-J-Eda^Ta-6J/J) were obtained from the Jackson Laboratory (JAX stock #000338). Msx1-deficient mice with a 129S4/SvJae genetic background were provided by the Mutant Mouse Resource and Research Centers (MMRRC stock #000068-UCD). We interbred heterozygous USAG-1 and Msx1 mice and analyzed the F₂ generation. To eliminate the influence of the mouse background, only F2 progeny USAG-1^−/−/Msx1^−/− mice were analyzed. Polymerase chain reaction was performed using KOD FX NEO polymerase (KFX-201; TOYOBO, Osaka, Japan) and specific primers. Embryos were obtained by timed mating; day E0 started from midnight, before finding a vaginal plug. Outbred pregnant ferrets were purchased from Marshall BioResources Japan Co. Ltd. A subgroup of the offspring was maintained in immunosuppressive condition, as previously reported (41 (link)).

Bmp7-LacZ knock-in (ICR) mice and Usag1-LacZ knock-in (C57BL/6) mice were produced as previously described [29 (link), 30 (link)]. Bmp7-deficient mice were embryonic lethal. Day E0 was established as midnight prior to finding a vaginal plug.
Polymerase chain reaction (PCR) amplification was performed using KOD FX NEO polymerase (TOYOBO, Osaka, Japan) and specific primers for genotyping.

cDNA was synthesized using Superscript IV (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Five hundred nanograms of the total RNAs extracted from the ASG, MSG_A, MSG_M, MSG_P, and PSG were used for the cDNA synthesis. KOD FX neo polymerase (Toyobo, Osaka, Japan) was used for RT-PCR. The PCR conditions were as follows: 95 °C for 1 min, followed by 22 cycles (for rp49) or 30 cycles (for TF genes) of 95 °C for 30 s, 58 °C for 30 s, followed by 68 °C for 1 min, and additional 68 °C for 1 min after the cyclic phase. The primer sequences are listed in Table S1.

cDNA was synthesized from 5 μg of RNA using 3′-Full RACE Core Set (Takara Bio) according to the manufacturer’s protocol. As the reverse transcription primer, ‘Oligo dT-3 sites Adapter Primer’ attached to the product was used. Coding sequence linked to mKGC was amplified using KOD FX neo polymerase (TOYOBO) (Tables 1 and 2). The PCR primer sequence is shown in Table 3.