Kapa express extract dna extraction kit

Genomic DNA from cells or tails of mice was extracted using a genomic extraction kit (Sigma, USA) or KAPA Express Extract DNA Extraction kit (Kapa Biosystems, USA). PCR analyses were performed using a standard protocol. To genotype MI-MAC Tc mice, hyg57s and hyg899As primers were used. To check site-specific recombination, pgk5 and CAG pro As primers were used. To check the insertion of the ELuc gene in the PhiC31 attachment site on the MI-MAC, ELuc128s and Eluc1116As primers, pgk5 and G418 As primers, pgk5 and CAG pro As primers, CAG pro S and R4 attP_Rv primers, and PhiC31 attL-Bfw and R4 attP_Rv primers were used (Fig. 3a). To genotype CAG-ELuc/MI-MAC Tc mice, ELuc128s and Eluc1116As primers were used. These primer sequences are listed in Supplementary Table 1.

For PCR and sequence analysis, genomic DNA was extracted from a tail biopsy with the KAPA Express Extract DNA Extraction Kit (Kapa Biosystems, London, UK). The sgRNA targeted region, 5′ genome-donor boundary, inside of knock-in donor, and donor-3′ genome boundary were PCR amplified. These PCR amplicons were then directly sequenced using the BigDye Terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Thermo Fisher Scientific). All primer sets used for genotyping analysis are shown in Supplemental Table 2.

Genomic DNA was extracted from the tail tip using the KAPA Express Extract DNA Extraction Kit (Kapa Biosystems, London, UK). For PCR and sequence analysis, we used external primers outside the HA, which amplied the targeted region (Additional file 18: Table S4). PCR was performed in a total volume of 15 μl under the following conditions: 1 cycle of 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 45 s; and 1 cycle of 72 °C for 3 min. The final reaction mixture contained 200 μM dNTPs, 1.0 mM MgCl₂, and 0.66 μM of primer. The PCR products were then directly sequenced using the BigDye Terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies). To confirm mosaic mutations, we sequenced individual TA clones in some cases.

Genomic DNA was extracted from the tail tips of 4-week-old rats using a KAPA Express Extract DNA Extraction Kit (Kapa Biosystems, London, UK). The genotyping primers for Il2rg were 5’-GACCAGAGGGGATTGCTGAG-3’ and 5’-GGTAGGTACCACATCTGCCC-3’; for Rag2, the genotyping primers were 5’-GGGGAGAAGGTGTCTTACGG-3’ and 5’-AGGTGGGAGGTAGCAGGAAT-3’. The PCR reaction mixture contained 200 μM dNTPs, 1.0 mM MgCl₂, and 0.66 μM of each primer in a total volume of 15 μl. The PCR cycles were as follows: one cycle at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 60°C for 1 min, and 72°C for 45 s. The PCR products were directly sequenced with BigDye Terminator v3.1 Cycle Sequencing Mix on an Applied Biosystems 3130 DNA Sequencer (Life Technologies), in accordance with the manufacturer’s standard procedure.

Previously, db, a mutation in the Lepr gene, was obtained using the PCR method with mismatched primers [28 (link), 29 (link)]. In the present study, for increased reliability, the PCR method without mismatched primers was established as follows. DNA was obtained from the tail or ear tissue from mice at 1 week of age, as described above, using the KAPA Express Extract DNA Extraction Kit (KAPA Biosystems, MA, USA). The DNA extracted was used as a template for the genes identified. The Lepr gene was amplified with forward and reverse primers (Supplementary Table 1) by PCR. The PCR product was digested with Hpy166II restriction enzyme (New England Biolabs Japan Inc.). The PCR products digested were analyzed by agarose gel (Kanto HC, Kanto Chemical Co., Inc., Tokyo, Japan) for identification of the three genotypes (+/+, db/+, and db/db). The Dock7 gene was amplified with one forward and two reverse primers (Supplementary Table 1). The PCR products were analyzed by agarose gel, and Dock7 genotyping (M/M, m/M, and m/m) was performed.

For every experiment, genomic DNA extraction was carried out on a pool of 15 injected, dechorionated embryos, using KAPA Express Extract DNA Extraction Kit (Kapa Biosystems, KK7103). Extraction mixes consisting of 15 embryos, 10 μl 10X Kapa Express Extract Buffer, 2 μl Express Extract Enzyme (1 U/μl) and 88 μl PCR grade water were incubated at 60 °C for 10 minutes and 95 °C for 5 minutes. The supernatant resulting from 1 minute full speed centrifugation was collected and stored at −20 °C.