Overnight cultures were diluted to an OD600 of 0.05 with fresh BHI or BHI with sCSP and incubated in an aerobic atmosphere containing 5% CO2 at 37°C for 2, 4, or 6 h. Total RNA was extracted and quantified by the method described above. We synthesized cDNA from 500 ng of total RNA with the PrimeScript™ RT reagent kit with a gDNA eraser (Takara). The primers used for RT‒PCR are shown in Table S1 . PCR was performed with Tks Gflex DNA polymerase (Takara) for 25 cycles. We used the lactate dehydrogenase gene ldh, a housekeeping gene in S. mutans, as an endogenous control (Merritt et al., 2005 (link)). PCR products were electrophoresed in a 1.5% agarose gel and stained with Midori Green Xtra (Nippon Genetics).
Midori green xtra
Manufactured by Nippon Genetics
Sourced in Germany, Japan
Midori Green Xtra is a nucleic acid stain used for the detection and visualization of DNA and RNA in agarose gels. It is a sensitive, non-toxic alternative to traditional ethidium bromide staining.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin rna 2 columns, by Macherey-Nagel (9 mentions)
M mlv reverse transcriptase rnase h minus, by Promega (9 mentions)
Gotaq polymerase, by Promega (9 mentions)
Random primer, by Promega (7 mentions)
Gotaq green master mix, by Promega (2 mentions)
Kapa hot start high fidelity polymerase, by Roche (2 mentions)
T7 endonuclease 1, by New England Biolabs (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Tks gflex dna polymerase, by Takara Bio (1 mentions)
Primescript rt reagent kit with a gdna eraser, by Takara Bio (1 mentions)
Nucleospin rna kit, by Takara Bio (1 mentions)
Agarose s, by Nippon Gene (1 mentions)
Fas 5 gel imaging system, by Nippon Genetics (1 mentions)
Q5 high fidelity polymerase, by New England Biolabs (1 mentions)
Quantaccuracy rt ramda cdna synthesis kit, by Toyobo (1 mentions)
Kod fx polymerase, by Toyobo (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Sitmem16a, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
20 protocols using midori green xtra
1
Quantitative RT-PCR of S. mutans Genes
2
Canine and Human Gene Expression Analysis
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Total RNA was extracted from the cultured cells using a NucleoSpin RNA kit (TaKaRa, Shiga, Japan) in accordance with the manufacturer’s instructions. cDNA was synthesized from 0.5 µg total RNA in accordance with the manufacturer’s instructions. The PCR amplification was performed using GoTaq Green Master Mix (Promega, Madison, WI, USA) based on the protocol provided by the manufacturer. The primer sequences were as follows: Canine CCND1: forward, 5′- GGTCTGCGAGGAGCAGAAGT-3′ and reverse, 5′- GATGAAGTCGTGTGGGGTCA-3′ (286-bp product size); canine TERT: forward, 5′- TTTGCAGACCTGCAGCCTTA-3′ and reverse, 5′- CACTGGCTGGTTGAATGGAA-3′ (780-bp product size); and human CDK4R24C: forward, 5′- AGTGGCTGAAATTGGTGTCG-3′ and reverse, 5′- ATGTGGCACAGACGTCCATC-3′ (218-bp product size). The PCR products were separated with 2% agarose gel electrophoresis and stained with Midori Green Xtra (NIPPON Genetics, Tokyo, Japan).
3
Amplification and Analysis of TNFα Locus
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A 1286 nt region of TNFα locus containing the target site, were PCR amplified using the following primer sets. The target locus was amplified for 35 cycles with specific forward (TNF-X-Fw:5’-CGCCACCACGCTCTTCTG-3’ ) and reverse (TNF-Alw-Rv:5’-CGGTTCAGCCACTGGAGC-3’ ) primers targeting exon 1 and exon 4 of the TNFα gene, respectively. The PCR reaction was performed using 50 ng of genomic DNA and Kapa Hot start high-fidelity polymerase (Kapa Biosystems, Wilmington, MA) in a high GC buffer according to the manufacturer’s protocol. The thermocycler setting consisted of one cycle of 95°C for 5 min, 30 cycles of 98°C for 20 s, 61°C for 15 s and 72°C for 30 s, and one cycle of 72°C for 1 min. The PCR products were analyzed on 1,5% agarose gel containing Midori Green Xtra (NIPPON Genetics Europe, Dueren, Germany). About 200 ng of PCR DNA was used for T7 endonuclease I and SmaI digestion analyses.
4
Cas12c RNA-Guided DNA Cleavage Assay
5
PCR Protocol using GoTaq Reagents
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PCR was performed using GoTaq Green Master Mix (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The sequences of the primers used for the evaluation are listed in Table S1 . PCR products were loaded on a 2% agarose gel (Agarose S: NIPPON GENE, Tokyo, Japan) with a fluorescent DNA staining reagent (Midori Green Xtra: NIPPON Genetics, Tokyo, Japan) and separated in 1× TAE buffer. Specific bands were detected by the FAS-V gel imaging system (NIPPON Genetics).
6
T7 Endonuclease I Assay for CRISPR Activity
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The widely used T7-endonuclease I assay targeted and digested hetero-duplexes formed by the hybridization of mutant and wild-type (wt) strands resulting in two smaller fragments; this method was performed to assess sgRNA-specific activity. After the transfection, the HEK293 cells were incubated for 48 hr. The cells were then pelleted, and the lysis performed using the QIAamp DNA Mini Kit (Qiagen). The PCR products were denatured and then reannealed using the following program: 95° C for 5 min, ramp down to 85°C at 2°C/s and ramp down to 25°C at 0.1 C/s. After the reannealing step and the consequent heteroduplex formation, 5 units of T7 endonuclease I (New England Biolabs, Ipswich, MA) were added to the mix and incubated 1 hr at 37°C. The product was resolved on 1,5% agarose gel containing Midori Green Xtra (NIPPON Genetics Europe, Dueren, Germany).
7
Quantitative RT-PCR of Airway Epithelial Cells
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For RT-PCR, total RNA from BCi-NS1 and CFBE human airway epithelial cells was isolated using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). Total RNA (0.5 µg/25 µL reaction) was reverse-transcribed using random primer (Promega, Mannheim, Germany) and M-MLV reverse transcriptase RNase H minus (Promega, Mannheim, Germany). Each RT-PCR reaction contained sense (0.5 µM) and antisense primer (0.5 µM) (Table 1 ), 0.5 µL cDNA and GoTaq polymerase (Promega, Mannheim, Germany). After 2 min at 95 °C, cDNA was amplified (targets of 35 cycles, reference GAPDH 25 cycles) for 30 s at 95 °C, 30 s at 56 °C and 1 min at 72 °C. PCR products were visualized by loading on Midori Green Xtra (Nippon Genetics Europe) containing agarose.
8
PCR Amplification with Q5 Polymerase
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PCR reactions were performed using Q5 High-Fidelity Polymerase (NEB) according to the manufacturer’s instructions with a final primer concentration of 0.5 μM (each) and 1.25 ng of diluted cDNA. The following settings were used for thermo-cycling: 30 s 98 °C, 30 cycles of 15 s of 98 °C, 20 s of 60 °C, 20 s of 72 °C followed by 2 min at 72 °C. PCR products were separated on 2% agarose gels and visualized by Midori Green Xtra (Nippon Genetics).
9
Quantifying TMEM16A and TMEM16F mRNA Expression in Mouse Colon
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For semi-quantitative RT-PCR of TMEM16F and TMEM16A, mRNA expression in proximal and distal segments of the mouse colonic epithelium, total RNA was isolated using NucleoSpin RNA II columns (Macherey-Nagel, Düren, Germany). Total RNA (1 µg/50 µL reaction) was reverse-transcribed using random primer (Promega, Mannheim, Germany) and M-MLV reverse transcriptase RNase H Minus (Promega, Mannheim, Germany). Each RT-PCR reaction contained sense and antisense primer for mouse TMEM16A and TMEM16F [39 (link)], 0.5 µL cDNA and GoTaq Polymerase (Promega, Mannheim, Germany). After 2 min at 95 °C cDNA was amplified 25 cycles for 30 s at 95 °C, 30 s at 56 °C and 1 min at 72 °C. PCR products were visualized by loading on Midori Green Xtra (NIPPON Genetics, Dueren, Germany) containing agarose gels and analyzed using ImageJ (NIH, Bethesda, MA, USA).
10
Cas12c Mediated In Vitro Plasmid Cleavage
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The Cas12c–guide RNA complex (1.25 μM) was prepared by mixing each purified Cas12c protein (Cas12c1, Cas12c2 and OspCas12c) (2.5 μM) with its cognate guide RNAs (crRNA and tracrRNA) (5 μM each) at 37°C for 5 min, in 5 μL buffer E. Each pre-assembled complex (2 μl, 1.25 μM, 250 nM final concentrations) was mixed with circular and linearized plasmid targets containing the 18-nucleotide target sequence and the TTTG PAM (8 μl, 100 ng), and then incubated at 37°C in a 10 μL reaction mixture (5 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl2, and 1 mM DTT) for 30 min. The reaction was stopped by the addition of Proteinase K (1 μl, 6.7 ng), and the products were resolved on a 0.8% agarose gel and then visualized with Midori Green Xtra (Nippon Genetics). Acidaminococcus sp. Cas12a (AsCas12a) and the Streptococcus pyogenes Cas9 (SpCas9) D10A mutant were prepared as described (Nishimasu et al., 2018 (link)), and the purified AsCas12a (TTTG PAM) and SpCas9 D10A (AGG PAM) were used as dsDNase and nickase controls, respectively. In vitro cleavage experiments were performed at least three times.
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