Genegreen nucleic acid dye

Methods of activity can be detected in three ways: (1) In vitro activity assays: the mole numbers (200 nM, 1 μl) of Cas9 proteins (Supplementary Table 2) are equal to that of sgRNA, and both cas9 protein and sgRNA were mixed and incubated in the buffer (20 mM HEPES, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA, 2 mM MgCl2, 5% glycerol, pH = 7.5) at 37°C for 30 min; the buffer was added with 100–150 ng target DNA, and incubated another 1.5 h at 37°; the cleavage products were detected by gel electrophoresis on 1% agarose gel stained with 1 × GeneGreen nucleic acid dye (TIANGEN). (2) Chemical rescue of His982 and His985 by imidazole: this process was similar to (1), with the only difference being the addition of a step: “the solution was mixed with 2 μl imidazole (500 mM) and incubated overnight at 37°C” before the detection of products. (3) In vitro detection of the off-target effects: the process was similar to (1), with the difference being sgRNA (continuous mutation with two bases as a unit in 20-nt sequence for target DNA) and the incubation time (4 h) after adding target DNA.

Normal and metastatic lymph nodes as well as Penl1 and CAF cells were screened for mycoplasma infection using the PCR Mycoplasma Test Kit (HuaAn Biotech, China). DNA from LNM, normal lymph nodes, Penl1 and CAF cells was also amplified and screened for HPV content through nested PCR using the GoTaq® Green Master Mix (Promega, USA) with consensus primers MY09/MY11 and GP5+/GP6+ [41 (link)]. Hep-2 and HeLa cells were analyzed in parallel as positive controls. All PCR products were electrophoretically separated on agarose gels, visualized using Tiangen GeneGreen nucleic acid dye (Tiangen, China), and imaged with the Gel Doc XRt System (BioRad, USA).
Genomic DNA of Penl1 was subjected to PCR amplification of fragments (containing exons 1–11 of TP53) suitable for sequencing [39 (link)]. PCR products were gel purified and sequenced from both ends in duplicate by Life technology.

Primer Premier 5 software (PREMIER Biosoft International, San Francisco, CA, USA) was used to design the primers (Table 1) for amplifying the bovine GHRHR gene (GenBank accession number: NC_037331.1). The fragments were amplified with 25 µL of reaction volume, which contained 1.0 μL of each forward and reverses primers (100 ng/μL), 1 µL genomic DNA (50 ng/µL), 12.5 uL of 2 × Taq Mix (TIANGEN, Beijing, China), and 9.5 µL double distilled water (ddH₂O). The PCR amplification was performed according to the procedure, including pre-denaturation at 94 °C for 5 min, 32 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 5 min. The PCR products of F1R1 were genotyped by PCR-RFLP. The digestion reaction mixture contained 5 uL PCR products, 1 uL 10 × buffer, 10 U restriction enzyme (PshAI, NEB, Ipswich, MA, USA), and sterile water. Samples were incubated at 37 °C for restriction enzyme overnight, and the digested products were analyzed by 1.5% agarose gel electrophoresis, stained with GeneGreen Nucleic Acid Dye (TIANGEN, Beijing, China) in 1% TBE. The PCR products of F2R2 were also electrophoresed on a 1.5% agarose gel, and then purified and sequenced through Sangon Biotech (Shanghai, China).

The wtSaCas9/SaCas9 mutant proteins (~100 nM) were first mixed with sgRNA (~200 nM) in the reaction buffer (20 mM Hepes, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA, 2 mM MgCl₂, 5% glycerol, pH 7.5) and incubated at 37 °C for 30 min. Then, the substrate DNA (~40 nM) was added to the Cas9-sgRNA solution and incubated at 37 °C for 1 h. Finally, the cleavage products were detected by gel electrophoresis on 1% agarose gel stained with GeneGreen nucleic acid dye (Tiangen Biotech Co. Ltd., Beijing, China). The activity was quantified using ImageJ, with the percentage of the cleavage rate being $100\times \left(1-\sqrt{1-fraction cleavaged}\right)$ [39 (link)].

The activity of DNase X was expressed by the DNA fragmentation assay. The DNA fragmentation was determined using plasmid analysis as described in Shiokawa’s report [22 (link)]. About 500 mg fresh sample tissue was minced into 300 μL extraction buffer (20 mM Tris-HCl pH 7.8, 1 mM β-mertoethyl alcohol, 300 mM NaCl, 3 mM MgCl₂, 0.5% Triton X-100, protease inhibitor), then homogenized and placed quietly on ice for 30 min. After centrifugation (10,000× g, 10 min, 4 °C), the supernatant was collected. The protein concentration in the supernatant was measured by the Bicinchoninic acid (BCA) kit (AR0197B, Boster, Wuhan, China). An amount of supernatant containing 4 μg protein was mixed into 10 μL reaction solution (50 mM HEPES-NaOH pH 7.2, 1 mM 2-mercaptoethanol) containing 500 ng supercoiled pBluescript II KS+, and incubated for digesting plasmid DNA at 37 °C for 10 min. After that, 10 μL digestion solution was loaded for electrophoresis by 1% agarose gel containing 1 × GeneGreen Nucleic Acid Dye (RT210, Tiangen Biotech, Beijing, China). The gel was visualized with a scanner (G: Box, GBOX Cambridge, UK). Quantification analysis of the band was performed using NIH Image J software (Media Cybernetics Inc., Rockville, MD, USA). The content proportion of DNA fragments below 1000 bp was calculated.

Total RNA was isolated from H9c2 cells using the E.Z.N.A.® Total RNA Kit (OMEGA, Norcross, GA, USA) according to the manufacturer's instructions. Reverse transcription to synthesize cDNA was performed using the PrimeScript^TM RT Reagent Kit with gDNA Eraser (Takara, Shiga, Japan). PCR amplification of the cDNA was performed using specific primers for rat KCNE1 (5’-TGTGGCAGGAAACAGATGAG-3’ and 5’-GGGTGAAGAAGCCGAAGAA-3’; 132 bp 56°C), Cx43 (5'-GTCTCACCTTTGTGCCTTCC-3' and 5'-GCTCACCTCCCTGATGCTAA-3'; 146 bp 56°C), BMP10 (5'-TGTCCATCCCTCACCACGAAGA-3' and 5’-TCCGTTGATACCAAGACCAGCA-3’; 175 bp 56°C), β-MHC (5’-CCCCTACGATTATGCGTTCTTC-3' and 5’-TCCTCCCTCTGCTTCTGTTTGA-3'; 195 bp 56°C) and β-actin (5’-CAACCTTCTTGCAGCTCCTCCGTC-3’ and 5’-TCTGACCCATACCCACCATCACACC-3’; 200 bp 56°C). PCR was performed in a Bio-Rad S1000^TM Thermal cycler (Bio-Rad, Hercules, CA, USA). cDNAs were amplified for 40 cycles. One round of amplification was performed at 95°C for 5 sec, at 56°C for 30 sec and at 72°C for 30 sec (Takara, Shiga, Japan). The PCR products (20 μl) were resolved on 2% agarose gels (Sigma, St. Louis, MO, USA) in 1× TAE buffer (0.04 M Tris acetate and 0.001 M EDTA), and GeneGreen Nucleic Acid Dye (TIANGEN, Beijng, China). The reaction products were visualized using a transilluminator (SYNGENE, Cambridge, UK) and a computer-assisted gel documentation system (SYNGENE, Cambridge, UK).

Based on the sequence of bovine GDF8 (GenBank accession number: NC_037329.1), two pairs of primers were designed for SNP scanning (Table 1). Five SNP sites were identified by PCR and sequencing, including two mutations in exon 1 (g.244C>G and g.400G>A), two mutations in exon 3 (g.5070C> A and g.5076T>C), and one mutation in the 3′untranslated region (UTR) (g.5148A>C). The PCR reactions were performed in a volume of 25 μL containing 20 ng of DNA template, 12.5 μL of 2×Taq Mix (including 0.1 U Taq polymerase/μL, 500 μM dNTP each, 20 mM Tris-HCl (pH8.3), 100 mM KCl, 3 mM MgCl₂, PCR reaction enhancer, optimizer, and stabilizer, TIANGEN, China), 1.0 μL of each primer (100 ng/μL), and sterile water. DNA fragments were amplified using the following parameters: initial denaturation at 95°C for 5 minutes, 32 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 2 minutes. These PCR products were electrophoresed on a 1.5% agarose gel stained with GeneGreen Nucleic Acid Dye (TIANGEN, China) in 1% Tris-borate-ethylenediaminetetraacetic acid buffer.