Ex taq

Degenerate primers G-D and G-1R (Additional file 6: Table S1) specifically targeting Class I and II PHA synthases [12 ] were used to amplify the phaC, phaC1 and phaC2 genes from the WGA seawater DNA. The PCRs contained 1× Ex Taq buffer, 1.5 mM MgCl₂, 200 μM dNTPs, 0.25 μM of each primer, 1% DMSO and 1 U of Ex Taq (TaKaRa, Japan). The PCR conditions were as follows: 94°C for 3 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 50 s; a final step at 72°C for 7 min. PCR products were run on an agarose gel and those within the range of 500 to 600 bp were excised from the gel, purified, and inserted into the pCR®4-TOPO® vector (Invitrogen, USA). The vectors were transformed into E. coli DH5α (TaKaRa, Japan) and selected on Lysogeny-Broth (LB) plates containing 50 μgmL⁻¹ kanamycin. Plasmids from the positive clones were isolated using PowerPrep™ Express Plasmid Purification Kits (ORIGEN, USA) and DNA sequencing was carried out at the RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Japan.

Adenovirus DNA in the recovered solution from dried spots on coverslips before and after nitrogen gas plasma treatment were extracted using the QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany). Reproducible extraction of adenovirus DNA by this method has been confirmed before and after capturing and concentrating adenovirus using magnetic anionic nanobeads [14 (link)]. Plasma-treated and untreated samples (20 μL) were solubilized in lysis buffer (560 μL Buffer AVL containing carrier RNA). The extracted DNA was bound to a column and then eluted in 60 μl of nuclease-free water. Viral DNA was amplified in a reaction mixture of 20 μL containing 2 μL of the eluted viral DNA as well as primers (0.2 μL each, 100 pmol/μL), Ex Taq (Takara Bio Inc.) (0.1 μL, 5 units/μL), and 10x Ex Taq buffer (2 μL), MgCl₂ (1.6 μL, 25 mM), dNTP mixture (1.6 μL, 2.5 mM each) and water (12.3 μL) under the following conditions: 30 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 2 min. PCR was carried out using the following primers specific for the adenovirus hexon gene;
Hexon-F: 5'-TGGGTGATAACCGTGTGCTA-3 ',
Hexon-R: 5'-TTAATGCTAGCCCCGTCAAC-3'
The PCR products were analysed by agarose gel electrophoresis on a 1.2% gel.

Primers were designed using Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). All primer sets are available upon request. PCR reactions contained 1 ul of LC Green Plus Melting Dye (BioFire Defense), 1 ul of Ex Taq Buffer, 0.8 ul of dNTP Mixture (2.5 mM each), 1 ul of each primer (10 uM), 0.05 ul of Ex Taq (Takara Bio Inc), 1 ul of gDNA, and water up to 10 ul. PCR was performed in an Eppendorf Mastercycler Pro S, using black/white 96 well plates (BioRad cat. No. HSP9665). PCR reaction protocol was 98°C for 30 sec, then 40 cycles of 98°C for 10 sec, 59°C for 20 sec, and 72°C for 15 sec, followed by 95°C for 20 sec and then rapid cooling to 4°C. Melting curves were generated with a Lightscanner HR 96 (Idaho Technology) over a 65–95°C range. Curves were analyzed with LightScanner Instrument and Analysis Software.

The sul1, sul2, sul3, sul4, dfrA1 (trimethoprim resistance), and IntI1 (class 1 integron integrase) among the 74 SMX-resistant isolates were detected using PCR. The dfrA1 gene plays a role in sulfonamide resistance in a different step of folate synthesis than sul (Huovinen et al., 1995 (link)). The IntI1 gene is frequently involved in the transfer of sul1 and sul2 (Koczura et al., 2016 (link)). The primers and cycle conditions are shown in Supplementary Table S2. PCR was carried out in a mixture of 0.025 U of Ex Taq (Takara, Japan), 0.5 μM each forward and reverse primer, 0.2 mM dNTP mixture (Takara, Japan), 1× Ex Taq buffer, and template DNA (50–100 ng). The amplification products were examined by electrophoresis on a 1.5% agarose gel with GelRed (Nacalai Tesque, Japan) staining. PCR product was confirmed based on band length and sequence.

Tissue samples were kept in CHAOS solution for at least a week to dissolve proteins at room temperature. Total DNA was extracted from the CHAOS solution with tissue samples by conventional phenol/chloroform extraction method. We used the primers reported by McFadden et al. (2006) (link) to amplify a fragment 5' end of the mitochondrial NADH-dehydrogenase subunit 2 gene (ND2) (16S647F: 5' -ACA CAG CTC GGT TTC TAT CTA CCA-3'; ND21418R: 5' -ACA TCG GGA GCC CAC ATA-3'). We also used two primers (1S: 5'-GGT ACC CTT TGT ACA CAC CGC CCG TCG CT-3'; 2SS: 5'-GCT TTG GGC GGC AGT CCC AAG CAA CCC GAC TC-3') (Wei et al. 2006 ) to amplify the internal transcribed spacer (ITS) of the nuclear ribosomal RNA gene. All PCR reactions contained 1 μL of DNA solution, 1.6 μL of 2.5 mM dNTP Mixture, 2 μL of 10X Ex Taq buffer, 2 μL of each 10 mM primer, Ex Taq (TaKaRa) 0.08 μL, and 11.32 μL of sterile distilled water. Amplifications of these markers were performed (GeneQ PCR Thermal Cycler) with the following thermal profile; 35 cycles of 90 sec at 94 °C, 60 sec at 58 °C, 60 sec at 72 °C. Amplified fragments were checked on 1% agarose gel electrophoresis. All the PCR products were subjected to digest excess primers and inactivation of dNTP using Exonuclease I (TaKaRa) and Shrimp Alkaline Phosphatase (TaKaRa). These DNA sequences were determined by ABI3000 using a research contract service (Ltd. FASMAC).

We used a glass capillary under a dissection microscope to pick up embryos. 4-cell mouse embryos were digested with protease to remove the zona pellucida, and then the embryos were transferred into 0.5% BSA/PBS and gently pipetted to separate individual blastomere. Finally, the individual blastomere was transferred into a PCR tube containing cell lysis buffer (YIKONGenomics). Each tube was centrifuge to facilitate the mix. The procedure of cell lysis and amplification was followed the manufacturer’s instructions. Then, the amplified products were used as templates in PCR analysis. PCR amplification made use of ExTaq (Takara) enzyme. ExTaq (Takara) was denature at 94 °C for 3 min, and PCR was performed for 34 cycles. The anneal temperature and extended time were changed with different genes. PCR products were performed electrophoresis and extracted by using QIAquick Gel Extraction Kit (Qiagen, USA).