Takara taqtm hot start version

Il1f10 (IL-38) deficient mice (GenBank accession number: NM_153077.2; Ensembl: ENSMUSG00000046845) were generated using CRISPR/Cas9 technology (Cyagen Biosciences, CA, USA) on a C57BL/6 background. Table 1 contains gRNA sequences used for the removal of Il1f10 exons 1-4 and nucleotides to confirm the deletion. Cas9 mRNA and gRNA were generated by in vitro transcription and injected into fertilized C57BL/6 eggs. Founders were genotyped by PCR using TaKaRa TaqTM Hot Start Version (Takara) and the PCR product was purified using the MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0, 9765 (Takara). The IL-38 deficiency was confirmed by PCR and gel electrophoresis, and DNA sequencing analysis (Figure 1). DNA sequencing revealed that F0 Mouse-ID #19, and F1 Mouse-ID #2 and #8 were missing 4725 bases in one Il1f10 allele, indicating loss of all Il1f10 exons. After transportation to our animal facility and further breeding, presence of homozygote IL-38 deficient knockout or wild type (WT) alleles were confirmed by ear clippings through Transnetyx prior to 3 weeks of age. All mice used in this study were between 8 and 10 weeks of age. All national and institutional guidelines for the care and use of laboratory animals were adhered to.

DNA was extracted by the same method as described above. The ESBL genotypes (TEM, SHV, CTX-M-1, CTX-M-2, and CTX-M-9) of the E. coli strains were determined by multiplex PCR as described elsewhere [49 (link),50 (link)].
The PCR reaction was conducted with TaKaRa Taq^TM Hot Start Version (Takara Bio, Inc.), which consists of Takara LA Taq polymerase plus a monoclonal antibody, and the TEM, SHV, CTX-M-1, CTX-M-2, and CTX-M-9 primers (Supplementary Table S2). Each 50-µL reaction consisted of 29.75 µL of sterilized distilled water, 0.25 µL of TaKaRa Taq^TM Hot Start polymerase, 5.0 µL of 10 × PCR buffer (Mg²⁺ plus), 4.0 µL of dNTPs, 1.0 µL of each primer, and 1 µL of the DNA template. The concentration of our primers was 10 μM per primer, with a final primer concentration of 0.2 μM in the 50 μL total volume of PCR reaction. The PCR reaction was performed using a SimpliAmp™ Thermal Cycler (Thermo Fisher Scientific) under the following reaction conditions: denaturation at 94 °C for 2 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1.5 min, and a final extension step at 72 °C for 5 min. The PCR products were separated in 1× TBE buffer by agarose gel electrophoresis as described above.