Neb buffer

To generate transgenic il1:gfp-t2a-il1-HA reporter fish, a fragment containing GFP and full-length of medaka il1 cDNA separated by t2a, a short viral sequences, were cloned into a vector containing 6.9 kb upstream region of the il1 gene (figure 2a). The plasmid at 10–25 ng µl⁻¹ concentration together with 1 µl I-SceI meganuclease and NEB buffer (NewEngland BioLabs) was co-injected into the blastomere at one-cell stage embryos. F0 larvae with GFP signal were selected for breeding.

Plasmids containing the target genes were cloned from the genomic DNA of S. noursei ATCC 11455 and amplified by PCR following 30 cycles of denaturing (95 °C for 30 s), annealing (65 °C for 30 s), and extending (72 °C, 1 min/kb) using high fidelity Phanta® DNA Polymerase. Amplification of target genes was confirmed by 1% agarose gel electrophoresis. The PCR products were purified using an Omega Biotech Gel Extraction Kit. pRSFDuet-1/pACYCDuet-1 vectors were digested in separate reactions containing 1× NEB buffer (New England Biolabs) with a selected pair of restriction enzymes for 3 h at 37 °C. The digested products were purified by agarose gel electrophoresis, and the target DNA fragments were extracted using an Omega Biotech Gel Extraction Kit. The resulting DNA products were ligated by homologous recombination at 37 °C for 0.5 h in 5 × CE II buffer with CE II enzyme. E. coli DH5α cells were transformed with 10 μL of the ligation product by heat shock. The resulting cells were plated on LB-kanamycin/chloramphenicol agar plates and grown for 15 h at 37 °C. Single colonies were picked and used to inoculate separate 5 mL cultures of LB-kanamycin/chloramphenicol medium. The cultures were grown at 37 °C for 12 h, and plasmids were isolated using an Omega Biotech Plasmid Mini Kit. The sequences of the resulting plasmids were confirmed by DNA sequencing.

To measure the efficiency of CCR5 genomic mutation, we performed T7 endonuclease 1 (T7E1) assay [79 (link)] and sequencing analysis as previously described [66 (link)]. Briefly, according to the protocol of Blood & Cell Culture DNA Midi kit (Tiangen, China), genomic DNA was extracted from the modified cells and PCR amplification of CCR5 gene with a set of primers (Additional file 6: Table S3). The PCR products were purified by Gel Extraction Kit (Promage). Then we used 300 ng purified PCR products combined with 2 µl 10 × NEB buffer (New England BioLabs) and appropriate deionized H₂O to make the final volume of 20 µl. The mixture was annealed to form the heteroduplexes and digested with five unites of mismatch-sensitive T7E1 (New England BioLabs) for 1 h at 37 °C [79 (link)]. The digested DNA was analyzed by 1.5% agarose gel and the editing frequency was quantified by Image J software as described previously [17 (link)]. The PCR products were also inserted into pEGM-T Easy Vector (Promega) for sequencing by a T7 primer.