Genextractor ta 100 automatic dna purification system

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.

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.