Applied biosystems 3130 dna sequencer

The expression level of GFP in embryos was measured using a BIOREVO immunofluorescence microscope (Keyence, Osaka, Japan). After incubation for 24 h, two-cell embryos were collected and genomic DNA was amplified using the GenomePlex Single Cell Whole Genome Amplification Kit (Sigma Aldrich, St Louis, MO, USA). After purification of the DNA, CRISPR-Cas-mediated mutations at target sites were analysed by direct sequencing.
For mutation detection, genomic DNA was extracted from tail biopsies using a GENEXTRACTOR TA-100 automatic DNA purification system (Takara Bio). The PCR products amplified with specific primer sets (Supplementary Table 6) were directly sequenced using the BigDye terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies).
Total RNA was extracted using Isogen reagent (Nippon Gene) from the spleen of 5-week-old rats. First strand cDNA was synthesized from 5 μg of total RNA that had been treated with DNase using an oligo(dT)12–18 primer and SuperscriptII reverse transcriptase (Invitrogen). PCR was performed with the primers for human SIRPA and rat Sirpa described in Supplementary Table 6.

Editing of the targeted gene was analysed using genomic DNA that was extracted from blood that was adhered to FTA cards using a GENEXTRACTOR TA-100 automatic DNA purification system (Takara Bio Inc., Shiga, Japan). 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 100 ng genomic DNA, 200 μM dNTPs, 1.0 mM MgCl₂, 0.66 μM each primer, and 0.4 U Taq DNA polymerase (Life Technologies Co.). To edit the cleavage site in the genome at the Il2rg locus, two primer sets were designed to amplify small (292 bp) and large (3,158 bp) fragments as shown in Supplementary Fig. S1. The PCR products were directly sequenced using the BigDye terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies Co.).

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 specific primers which could amplify the targeted region. PCR was performed in a total volume of 15 μl under the following conditions; 1 cycle of 94 °C for 1 min, 30 cycles of 98 °C for 10 s, 60 °C for 15 s and 68 °C for 30 s. 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).

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.

A pair of TALENs targeting exon 1 of rat Scn1a (Ensembl: ENSRNOG00000053122) was designed and constructed using a two-step assembly method with a Platinum Gate kit as previously reported (22 (link)). Assembled sequence was 5′-TGCAGGATGACAAGATGgagcaaacagtgcttGTACCACCAGGACCTGA-3′, where uppercase and lowercase letters indicate TALEN target sequences and spacer sequence, respectively. TALENs were microinjected into fertilized eggs of Fisher 344 (F344) rats and transferred into the oviducts of pseudopregnant female Wistar rats, as previously described (23 (link)). Genomic DNA was extracted from the tail using the GENEXTRACTOR TA-100 automatic DNA purification system (Takara Bio) and amplified with specific primer sets (forward 5′-TCCTCACTTGTTGGGTCTCA-3′, reverse 5′-TCAGGGTGACTTCAGCATTTC-3′). The polymerase chain reaction (PCR) products were directly sequenced using the BigDye terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies). Rats from the fifth generation or later were used in all experiments. Genotypes were assessed by running the PCR products from ear DNA on a Caliper electrophoresis system at postnatal day (P9). Only male rats were used in experiments to eliminate sex differences.

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.