Intestinal contents were collected from piglets with watery diarrhea and vomiting in the western provinces of China (Gansu, Qinghai, Ningxia, Shanxi, and Chongqing) between 2015 and 2018. The contents were diluted with phosphate-buffered saline (PBS) at a ratio of 1:100 for RNA extraction with RNAiso Plus (Takara, Japan) according to the manufacturer’s instructions. The extracted RNA was then reverse transcribed using HiScript II RT SuperMix (Vazyme, China). Briefly, a mixture of 4 μL of gDNA wiper Mix, 1 μg of RNA, and nuclease‐free water to 16 μl was incubated at 42°C for 2 min, after which 5 μl of HiScript II RT SuperMix was added and the mixture was incubated at 50°C for 15 min and 85°C for 5 s. Detection of pathogens was performed by a multiplex PCR method developed previously in our laboratory [21 (link)]. Briefly, 5 μl of cDNA was used as a template and mixed with the specific primer pair, 12.5 μl of 2× PCR master mix (Vazyme, China), and nuclease‐free water to 25 μl. The reaction was carried out under the following conditions: 95 °C for 3 min, followed by 35 cycles of 15 s at 95°C, 15 s at 52°C, and 30 s at 72°C, and final extension at 72°C for 5 min. The PCR products were then analyzed by agarose gel electrophoresis.
Pcr master mix
Manufactured by Vazyme
Sourced in China
The PCR master mix is a ready-to-use solution containing all the necessary components for performing polymerase chain reaction (PCR) amplification, including DNA polymerase, dNTPs, and buffer. It is designed to simplify the PCR setup process and ensure consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Acer qpcr sybr green master mix, by Vazyme (2 mentions)
Hiscript 2 rt supermix, by Vazyme (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Takara reverse transcription kit, by Takara Bio (1 mentions)
Cfx maestro, by Bio-Rad (1 mentions)
Atcc scrc 1041, by American Type Culture Collection (1 mentions)
Atcc htb 26, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Sybr green master mix, by Vazyme (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dna isolation mini kit, by Vazyme (1 mentions)
Electrophoresis reagents, by Bio-Rad (1 mentions)
Fastpure plasmid mini kit, by Vazyme (1 mentions)
Fastpure gel dna extraction mini kit, by Vazyme (1 mentions)
Applied biosystems 2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Nuclease free water, by Tiangen Biotech (1 mentions)
Dna extraction kit, by Tiangen Biotech (1 mentions)
13 protocols using pcr master mix
1
Multiplex PCR Detection of Enteric Pathogens
2
Fungal DNA Extraction and Gene Amplification
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Genomic DNA of ten representative fungal isolates was extracted using the CTAB method as described previously [38 ]. Mycelium was harvested from the colony surface by a sterile medicine spoon. The TEF1 and RPB2 genes were amplified using the primer pairs EF-1/EF-2 (5′-ATGGGTAAGGARGACAAGAC-3′/5′-GGARGTACCAGTSATCAT G-3′) and 7cf/11ar (5′-CCCATRGCTTGYTTRCCCAT-3′/5′-GCRTGGATCTTRTCRTCSA CC-3′) [39 (link),40 (link)]. Polymerase chain reaction (PCR) amplification was conducted in 50 μL volume reaction system containing 25 μL of 2 × PCR Master Mix (Vazyme, Nanjing, China), 2 μL of each primer (10 μM), 2 μL of genomic DNA template and 19 μL of sterile distilled water. PCR was performed with the thermal cycling parameters of 95 °C for 3 min, followed by 34 cycles of denaturation at 95 °C for 30 s, annealing at 54–57 °C for 30 s, extension at 72 °C for 1 min and final extension at 72 °C for 8 min. The PCR products were visualized by running with 1.0% agarose gel and staining with GoldView™. The PCR products were sequenced using the dideoxy termination method in Shanghai Sangon Company in China.
3
CRE Fragment Amplification and Sanger Sequencing
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Genomic DNA was extracted from human peripheral blood using an extraction kit from Tiangen (Beijing, China). The CRE fragment was amplified by polymerase chain reaction (PCR) in 20 μl of reaction mixture that contained 2 μl DNA template, 10 μl 2 × PCR Master Mix (Vazyme, Nanjing, China), and 1 μl of each primer. The primer pair was 5'-GGG AGG GAA CTA TGG AGG CAT C-3' and 5'-GGC CTA GCA GGG CGA GAA TAG-3'. The PCR condition was initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 5 min. Variants were identified by bidirectional Sanger sequencing (BGI, Shanghai, China).
4
Quantification of Immune Checkpoint Genes
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For RT-qPCR, a total of 1 μg of RNA was extracted using TRIzol reagents (Thermo Fisher Scientific (Waltham, MA USA), Cat#15596026) and employed for the reverse transcription reaction, utilizing a TAKARA reverse transcription kit (Takara, Beijing, China), following the manufacturer’s instructions. Following that, a PCR master mix from Vazyme (Nanjing, China) was used, and the detection was conducted using Bio-Rad CFX Maestro (Hercules, CA, USA). The primer sequences employed were as follows:
CD276-F: 5′-CTGGCTTTCGTGTGCTGGAGAA-3′;
CD276-R:5′-GCTGTCAGAGTGTTTCAGAGGC-3′;
CD252 (TNFSF4)-F: 5′-CCTACATCTGCCTGCACTTCTC-3′;
CD252 (TNFSF4)-R: 5′-TGATGACTGAGTTGTTCTGCACC-3′;
TNFSF9-F: 5′-GGCTGGAGTCTACTATGTCTTCT-3′;
TNFSF9-R: 5′-CGTGTCCTCTTTGTAGCTCAGG-3′;
CD70-F: 5′-GCTTTGGTCCCATTGGTCG-3′;
CD70-R: 5′-CGTCCCACCCAAGTGACTC-3′;
CD274 (PD-L1)-F: 5′-TGGCATTTGCTGAACGCATTT-3′;
CD274 (PD-L1)-R: 5′-TGCAGCCAGGTCTAATTGTTTT-3′;
GAPDH-F: 5′-CTGGGCTACACTGAGCACC-3′;
GAPDH-R: 5′-AAGTGGTCGTTGAG GGCAATG-3′.
5
Evaluating Cytotoxicity and Gene Expression
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Polysorbate 20 and 80, Tween 80 (polyethylene glycol sorbitan monooleate), polyethylene glycol (PEG), DMEM culture medium trypsin, MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and Fetal Bovine Serum (FBS), were purchased from (Merck, Darmstadt, Germany). Cancer cell lines (MDA-MB-231, ATCC HTB 26) and human fibroblasts foreskin (ATCC SCRC-1041) as normal cell line were obtained from the Pasteur Institute of Iran. The PCR Master Mix and SYBR Green Master mix were purchased from Vazyme Biotech (Nanjing, China). The RNA extraction kit and cDNA Synthesis Kit were purchased from CinnaGene (Tehran, Iran).
6
MALAT1 Expression Quantification
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Total RNA was extracted from cells or mouse lung tissue using TRIzol reagent according to manufacturer’s instructions (Invitrogen, New York, United States). RNA was reverse-transcribed to complementary DNA (cDNA) using PCR Master Mix (Vazyme Biotech, Jiangsu, China). Real-time quantitative PCR was performed using an AceR qPCR SYBR Green Master Mix (Vazyme Biotech, Jiangsu, China). The expression of MALAT1 was normalized to GAPDH and calculated using the 2–ΔΔCt method. The primers used for qRT-PCR are listed in Table 3 .
7
Measuring Mitochondrial DNA Copy Number
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For this step, we followed the method referred to in our previous research [43 (link)]. Total cellular DNA was extracted using the DNA Isolation Mini Kit (DC102-01, Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions and used for the detection of mtDNA copy number by qPCR with the following reagents: TaqMan Universal PCR master mix and the primers (16S rRNA and 18S rRNA). MtDNA levels were assessed using the mitochondrial genes 16S rRNA; nuclear 18S rRNA served as a loading control.
8
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from cells using Trizol reagent according to manufacturers' instructions (Ambion, Austin, USA). Then RNA was reverse translated to complementary DNA using the PCR Master Mix (Vazyme Biotech, Nanjing, China). qRT-PCR was performed using an AceR qPCR SYBR Green Master Mix (Vazyme Biotech) according to the manufacturer's instructions. The expression of NKILA was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNA expression and calculated using the 2−ΔΔCt method. The primers used for qRT-PCR are listed in Table 3 .
9
Recombinant protein expression in E. coli
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Escherichia coli DH5α and Rosetta (DE3) were obtained from the Stratagene, USA and used as the host strains for DNA cloning and expression, respectively. They were routinely cultured in Luria-Bertani (LB) medium containing Kanamycin (50 μg/mL) in a rotary shaker at 37°C and 200 rpm. The plasmid pANY1 was obtained from the Shenyang Agricultural University, China (Gao et al., 2018 (link); Liu et al., 2018 (link)), and was used as the expression vector.
The PCR master mix, restriction endonuclease One-Step Cloning Kit, Fast Pure Plasmid Mini Kit and Fast Pure Gel DNA Extraction Mini Kit were purchased from the Vazyme (Nanjing, China). The Ni2+-NTA resin for protein purification, were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA, USA). Isopropyl β-D -1-thiogalactopyranoside (IPTG), analytical and HPLC grade chemicals used for enzyme assays and characterization were obtained from the Aladdin (Shanghai, China) and the Sinopharm Chemical Reagent (Beijing, China).
The PCR master mix, restriction endonuclease One-Step Cloning Kit, Fast Pure Plasmid Mini Kit and Fast Pure Gel DNA Extraction Mini Kit were purchased from the Vazyme (Nanjing, China). The Ni2+-NTA resin for protein purification, were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA, USA). Isopropyl β-
10
Carbapenemase Gene Detection by PCR
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DNA was extracted from purified bacterial isolates using a TIANamp Bacteria DNA Kit (Tiagen, China) and tested by PCR with primers specific to five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaOXA-48, blaIMP). A volume of 12.5μL of PCR Master Mix (Vazyme, USA) was mixed with 2μL of forward and reverse primers in a 25μL reaction. Reactions were amplified on the Applied Biosystems 2720 Thermal Cycler (ThermoFisher Scientific, USA) using following the cycling conditions: an initial 94℃ 2 min hold, followed by 36 cycles at 94℃ for 30s, 60℃ for 40s and 72℃ for 1 min, followed 72℃ for 5 min. The appropriately sized PCR products were confirmed by DNA sequence analysis.16 (link) A positive result means that at least one carbapenemase gene was detected in this specimen. The negative result means that there were no blaKPC, blaNDM, blaVIM, blaOXA-48 or blaIMP carbapenemase genes detected by the PCR-based DNA sequence analysis. K. pneumoniae ATCC 2146 (blaNDM positive) and K. pneumoniae ATCC BAA-1705 (blaKPC positive) were used as positive controls. E. coli ATCC 25922 was used as a negative control.
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