The polymorphic variants mentioned in Table 1 were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). PCR amplification was carried out in a final volume of 25 µL using a DNA Engine Dyad Thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The reaction mixture consisted of 100 ng of genomic DNA, 0.25 µM of each primer (TiBMolBiol, Berlin, Germany), 1 U of Taq DNA polymerase and respective buffer (DreamTaq Green DNA polymerase, Thermo Fisher Scientific, Waltham, MA, USA), 200 µM dNTP Mix (Thermo Fisher Scientific, Waltham, MA, USA) and deionized water. PCR reaction conditions were optimized for each polymorphism. Characteristics of the polymorphisms and the specific primer sequences are shown in Table 2 . Amplified fragments were then digested with the appropriate restriction enzyme (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions, and visualized after electrophoresis on 2% or 3% agarose gel with Midori Green Advanced DNA Stain (Nippon Genetics, Europe GmbH, Düren, Germany).
Midori green advanced dna stain
Manufactured by Nippon Genetics
Sourced in Germany
Midori Green Advanced DNA Stain is a DNA-binding dye used for the detection and visualization of nucleic acids in various applications, such as gel electrophoresis, real-time PCR, and fluorescence microscopy. It is a sensitive and versatile stain that can bind to both double-stranded and single-stranded DNA, as well as RNA, emitting a bright green fluorescence upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
Dntp mix, by Thermo Fisher Scientific (1 mentions)
Dreamtaq green dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dna engine dyad thermocycler, by Bio-Rad (1 mentions)
Bsuri, by Thermo Fisher Scientific (1 mentions)
Hotstarttaq plus dna polymerase kit, by Qiagen (1 mentions)
Hotstarttaq plus master mix kit, by Qiagen (1 mentions)
Dna blood mini kit, by Qiagen (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Sm 2000r microtome, by Leica (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
9 protocols using midori green advanced dna stain
1
Genotyping of Polymorphic Variants
2
Genetic Polymorphism Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Basic information on selected polymorphic variants are presented in Table 8 . Genotyping was performed in the Molecular Biology Laboratory of Poznan Medical Science University via polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). The primers published by Frosst et al. [89 (link)], Hormon et al. [90 (link)], and Hol et al. [91 (link)] and the restriction enzymes HinfI (EURx, Gdansk, Poland), BsuRI (Thermo Fisher Scientific, Waltham, MA, USA), and MspI (EURx, Gdansk, Poland) for MTHFR (rs1801133), MTR (rs1805087) and MTHFD1 (rs2236225), respectively, were used. Products were analyzed via electrophoresis on 2% agarose gel with Midori Green Advanced DNA Stain (Nippon Genetics, Düren, Germany). Positive and negative controls were included in each reaction as quality control, and for accuracy of genotyping, 90% of samples were repeated. The concordance between the original and the duplicate samples for all the analyzed SNVs was 100%.
3
Isolation and Analysis of Nuclear DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Low-molecular-weight nuclear DNA was isolated from approximately 106 cells as described by Hinshaw et al. [19 (link)], with slight modifications. Briefly, 106 mock-infected or SADS-CoV-infected cells were harvested. The cells were washed in PBS and then resuspended in 500 μL of ice-cold lysis buffer (10 mM Tris [pH 7.5], 1 mM EDTA, 0.2% Triton X-100) containing 500μg/mL protease K for 8−10 h at 55°C. After incubation on ice for 20 min, the lysates were centrifuged at 12,000 g at 4°C for 30 min, and the supernatants were extracted with buffered phenol, then with buffered phenol–chloroform, and finally with chloroform-isoamyl alcohol (24:1, vol/vol). DNA was ethanol precipitated with 500 mM NaCl. DNA samples were resuspended in 20 μL of distilled water and treated for 60 min at 37°C with ribonuclease at a final concentration of 20 μg/mL. One-third of the DNA sample was analyzed on a 1.5% agarose gel containing Midori Green Advanced DNA Stain (NIPPON Genetics) in 1 × Tris-borate-EDTA buffer, and the sizes of the oligonucleosomal DNA fragments were estimated using 2-kb markers.
4
Genotyping Angiogenesis-Related SNPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five SNPs, localized in the genes encoding angiogenesis-related factors, were selected according to the SNP database (dbSNP) of the National Center for Biotechnology Information (NCBI) [41 ] (http://www.ncbi.nlm.nih.gov/projects/SNP , accessed on 22 February 2022) and the 1000 Genomes Project data (http://www.internationalgenome.org/ , accessed on 22 February 2022), based on minor allele frequency (MAF) of at least 5% in European populations. Basic information about the tested variants is presented in Table 7 .
Genotyping was performed in the Molecular Biology Laboratory of Poznan University of Medical Science by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).
The primers and restriction enzymes used for the RFLP reactions were from previously published research and are presented inTable 2 [29 (link),40 (link),73 (link),74 (link)]. Products were analyzed by electrophoresis on 2% agarose gel with Midori Green Advanced DNA Stain (Nippon Genetics, Düren, Germany). Positive and negative controls were included in each reaction and for quality control, 10% of the samples were randomly genotyped twice by different individuals, and the reproducibility was 100%. SNP characteristics, primer sequences, and details of the PCR-RFLP assays are presented in Table 8 .
Genotyping was performed in the Molecular Biology Laboratory of Poznan University of Medical Science by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).
The primers and restriction enzymes used for the RFLP reactions were from previously published research and are presented in
5
Multiplex PCR Serotyping of E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The STEC/AE-STEC/EPEC serotypes were determined by the PCR assay based on the EU-RL protocol for E. coli (EU-RL VTEC_Method 003) [24 ,25 (link)], for O-antigen-encoding genes (wzx)—O26, O103, O111, O145 and O157—and the protocols proposed by Durso et al., Gannon et al. and Mora et al. [26 (link),27 (link),28 (link)] for H antigens encoding the fliC gene (specific to flagellar genes)—H7, H8, H11, H21 and H28. All PCR amplifications were performed with the HotStartTaq Plus DNA Polymerase Kit (Qiagen, Venlo, Netherlands) and the HotStartTaq Plus Master Mix Kit (Qiagen), according to the manufacturer’s recommendations. The annealing temperature for every primer was described previously [15 (link)]. The PCR products were separated by electrophoresis in 2% agarose gel with the Midori Green Advanced DNA Stain (Nippon Genetics Europe GmbH, Düren, Germany).
6
Analysis of PDCoV Infection in ST Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ST cells were grown at 3.5 × 105 cells/well in 6-well tissue culture plates for 1 day and then mock infected or infected with PDCoV at a multiplicity of infection (MOI) of 1. In addition, cells were pretreated with Z-VAD-FMK or CsA for 1 h followed by PDCoV infection. At the indicated times, cells were harvested, washed with PBS, and then incubated in a cell lysis buffer (10 mM Tris, pH 7.5, 1 mM EDTA, and 0.2% Triton X-100) containing 500 μg/ml protease K for 24 h at 55 °C. The DNA was then extracted twice with phenol/chloroform, precipitated with isopropanol, and resuspended in distilled water. Next, the purified DNA was incubated with 20 μg/ml ribonuclease A for 1 h at 37 °C, electrophoresed on a 1.2% agarose gel containing Midori Green Advanced DNA Stain (NIPPON Genetics, Tokyo, Japan), and photographed.
7
PEDV Infection and Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vero cells were grown at 3.5×105 cells/well in 6-well tissue culture plates for 1 day and then mock infected or infected with PEDV at a multiplicity of infection (MOI) of 0.1. In addition, cells were pretreated with Z-VAD-FMK or CsA for 1 h followed by PEDV infection. At the indicated times, cells were harvested, washed with PBS, and then incubated in a cell lysis buffer (10 mM Tris, pH 7.5, 1 mM EDTA, and 0.2% Triton X-100) containing 500 μg/ml protease K for 24 h at 55 °C. The DNA was then extracted twice with phenol/chloroform, precipitated with isopropanol, and resuspended in distilled water. Next, the purified DNA was incubated with 20 μg/ml ribonuclease A for 1 h at 37 °C, electrophoresed on a 1.2% agarose gel containing Midori Green Advanced DNA Stain (NIPPON Genetics), and photographed.
8
VDR Gene SNPs Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We examined four SNPs (rs7975232, rs1544410, rs228570, rs731236) in the VDR gene, and all of the SNPs had minor allele frequencies (MAF) greater than 5%. Traditionally these allelic variants have been designated by the upper and lower case of the starting initial of the named loci, e.g. BsmI (b and B), TaqI (t and T), ApaI (a and A) and FokI (f and F). Genomic DNA was isolated from whole blood collected in K3-EDTA tubes using the Qiagen DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer's instructions and was stored at minus 80°C. A NanoDrop 2000 spectrophotometer (Wilmington, DE, USA) was used to evaluate the quality and quantity of DNA. Genotyping was performed in the Molecular Biology Laboratory of Poznan Medical Science University using the PCR/RFLP method as described previously [23] [24] (link)[25] (link). Products were analyzed by electrophoresis on 2.5% agarose gel with Midori Green Advanced DNA Stain (Nippon Genetics, Europe GmbH) (Tab. 1).
9
DNA Extraction from FFPE Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From the formalin-fixed paraffin-embedded (FFPE) tissue specimens, 10 μm flakes were cut with a Leica SM2000R microtome (Leica Microsystems GmbH, Wetzlar, Germany). Macrodissection was used when applicable to increase the yield of tumor DNA. Two to fifteen tissue flakes were deparaffinized and genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Venlo, the Netherlands) as previously reported [9 (link)]. DNA quality was inspected with NanoDrop spectrophotometer (Thermo Fischer Scientific, Waltham, MA) and agarose gel electrophoresis (with Midori Green Advanced DNA Stain; Nippon Genetics EUROPE GmbH, Dueren, Germany), and DNA concentration was determined using Qubit dsDNA HS Assay Kit and Qubit 2.0 Fluorometer (Molecular Probes/Life Technologies, Paisley, UK).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!