The 234 S. aureus strains isolated and the strain ATCC29213 were removed from the −80°C refrigerator and screened using CHROMagar™ Staph aureus (CHRO Magar, France). Single purple-red colonies were selected for purification and culture. DNA was extracted using the TIANamp Bacteria DNA Kit (Tiangen Biotech Co., Ltd., China). Bacterial genomic DNA was used as the template, and hla primers (F: 5′-GGTTTAGCCTGGCCTTC-3′; R: 5′-CATCACGAACTCGTTCG-3′), 2× Taq PCR Master Mix (Vazyme Biotech Co., Ltd., China), and deionized water (Tiangen Biotech Co., Ltd., China) to prepare a 20-μL reaction system. Gene fragment amplification was performed at 58°C, used as the optimum annealing temperature. The sequencing results were uploaded to the NCBI website for sequence comparison by using the BLAST tool.
Taq pcr master mix
Manufactured by Vazyme
Sourced in China
2× Taq PCR Master Mix is a ready-to-use solution containing Taq DNA polymerase, dNTPs, and reaction buffer optimized for standard PCR amplification. It is designed to simplify PCR setup and improve reproducibility.
Automatically generated - may contain errors
Lab products found in correlation
T100 thermal cycler, by Vazyme (2 mentions)
Staph aureus, by CHROMagar (1 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (1 mentions)
Axyprep multisource rna miniprep kit, by Corning (1 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Hiscript 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Simpliamp, by Thermo Fisher Scientific (1 mentions)
Pct 200 peltier thermal cycler, by Bio-Rad (1 mentions)
Seqman, by DNASTAR (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 kit, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
S1000 thermal cycler, by Bio-Rad (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Pbs buffer, by Sangon (1 mentions)
Tbe buffer, by Sangon (1 mentions)
Rnase free ddh2o, by Sangon (1 mentions)
El tmb chromogenic reagent kit, by Sangon (1 mentions)
Tween 20, by Sangon (1 mentions)
Minibest whole blood genomic dna extraction kit, by Takara Bio (1 mentions)
19 protocols using taq pcr master mix
1
Screening and Sequencing of S. aureus hla Gene
2
Viral Detection in Chicken Liver Tissues
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The total RNA was extracted from the liver tissue sample suspension using the AxyPrep Multisource RNA Miniprep Kit (Axygen, USA) and cDNA was synthesized using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). For detection of ARV, the PCR was performed with specific primers of S2 gene listed in Table 1 (26 (link)). The PCR reaction volume was 25 μl containing 12.5 μl of 2× Taq PCR Master Mix (Vazyme, China), 1 μl of each primer, 9.5 μl of double-distilled water (ddH2O), and 1 μl of the cDNA template. The PCR cycling conditions for the S2 gene amplifications were as follows: 1 cycle of 94°C for 5 min, 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, followed by a final extension step of 72°C for 10 min. In addition, PCR detection for chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), A subgroup of avian leukosis virus (ALV-A), B subgroup of avian leukosis virus (ALV-B), J subgroup of avian leukosis virus (ALV-J), and K subgroup of avian leukosis virus (ALV-K) was performed. The primers used are listed in Table 2 .
3
Genotyping of Transgenic Mice
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A small piece of the ear was cut, and DNA was extracted with the phenol‐chloroform method. PCR was performed to identify WT or mutant mice. For PCR amplification, ≈500 ng DNA was used in a 30 µL reaction volume containing 15 µL 2× Taq PCR MasterMix (Vazyme) and 1 µm primers. The sequences of the primers are listed in Table S1 of the Supporting Information. The extracted DNA and primers were denatured initially at 94 °C for 4 min followed by 25 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 50 s, and a final extension at 72 °C for 5 min. Amplicons were separated using 1.5% agarose gel, stained with GelRed, and photographed with GelDoc‐It Imaging System (UVP).
4
Verification of novel miRNA via stem-loop qRT-PCR
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To further verify our identification results, 19 randomly chosen sequences, including all 9 novel miRNAs as well as 10 conserved ones, were subjected to stem-loop qRT PCR75 . Stem-loop qRT-PCR amplifications were carried out using universal primer, RT primer and forward primer. In the first step, reverse transcription was performed using a HiScript 1st Strand cDNA Synthesis kit (Vazyme Biotech, NY, USA) in 20-μL reaction volumes consisting of 1 μg RNA, 0.5 μL RT primer, 5 μL of 2× RT Mix, 1 μL RT Enzyme Mix and RNase-free double-distilled water. Reaction conditions were 25 °C for 5 min, 42 °C for 20 min, 85 °C for 10 min and 4 °C for 5 min. In the second step, QRT-PCR was carried out in reaction mixtures comprising 1 μL cDNA, 0.5 μL universal primer, 0.5 μL forward primer, 5 μL of 2× Taq PCR Master Mix (Vazyme Biotech) and 3 μL distilled water. The qRT-PCR protocol was as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 15 s, with a final step of 72 °C for 5 min and then 4 °C for 2 min. The resulting PCR products were detected by 1.5% agarose gel electrophoresis.
5
Genotyping of PHS Resistance Alleles
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The alleles of 10 genes associated with PHS resistance were determined using the functional markers as described in Table S2 . For the sequence tagged site (STS) and cleaved amplified polymorphic sequence (CAPS) markers, a 20 μL reaction mixture was prepared by mixing 1 μL of 50–100 ng μL−1 template DNA, 1 μL each of the forward and reverse primers (10 μM), 10 μL 2 × Taq PCR Master Mix (P111-03, Nanjing Vazyme Biotech Co. Ltd., Nanjing, China), and 7 μL of sterilize ddH2O. Amplification of DNA was performed in a SimpliAmp thermal cycler (Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China). The Kompetitive Allele-Specific PCR (KASP) assays were carried out in a 5 μL reaction mixture including 2.5 μL PARMS SNP master mix (GTE001-2, Wuhan Genetides Biotech Co., Ltd., Wuhan, China), 0.056 μL primer mix, 0.04 μL Mg2+, 2.2 μL template DNA (20–50 ng μL−1) and 0.204 μL ddH2O using a BIO-RAD S1000 Thermal Cycler PCR System (Bio-Rad Laboratory Inc., Hercules, CA, USA). Genotyping of KASP markers was performed following the protocol as described by Rasheed et al. [33 (link)].
6
Multiplex gene expression analysis
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PCR amplification of HER-2, E-cadherin, N-cadherin, Vimentin, CEA, CA15-3, SF and reference genes was performed using the PCT-200 Peltier thermal cycler (MJ Research, Waltham, MA, USA). PCR was performed in a 25-μL reaction volume that contained 12.5 μL 2×Taq PCR Master Mix (P112-01, Vazyme), 3 μL cDNA template, 0.5 μL of each primer and 8.5 μL deionized water. The PCR parameters were 94 °C for 2 min; followed by 35 cycles of 94 °C 30 s, 58 °C 30 s and 72 °C 15 s, with a final extension at 72 °C for 10 min. Amplification products were visualized via 1.2% agarose gel electrophoresis (1645052, 1704486, Bio-Rad, Hercules, CA, USA).
7
Fungal DNA Extraction and Sequencing
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Total genomic DNAs were extracted from fresh fungal mycelia grown on potato dextrose agar (PDA; DifcoTM, Becton, Dickinson and Company, Sparks, MD, USA) using the cetyltrimethylammonium bromide (CTAB) method (Porebski et al. 1997 ) and stored at -20 °C until polymerase chain reaction (PCR). PCR amplifications were performed in a reaction mixture consisting of 12.5 μL 2 × Taq PCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 1 μL each of 10 μM primers, 2 μL of the undiluted genomic DNA, adjusted to a final volume of 25 μL with distilled deionised water. Seven loci, including ITS (White et al. 1990 ), tef1 (O’Donnell et al. 1998b ), CaM (O’Donnell et al. 2000b ), rpb1 (O’Donnell et al. 2010 (link)), rpb2 (Liu et al. 1999 (link); Reeb et al. 2004 (link)), tub2 (O’Donnell & Cigelnik 1997 (link)), and H3 (Roux et al. 2001 ) were amplified and sequenced. The PCR primer pairs and amplification conditions are listed in Table 1 . The PCR products were visualised using 1 % agarose electrophoresis gels. Sequencing was done by the Tianyi Huiyuan Company (Beijing, China) and the SinoGenoMax Company (Beijing, China).
8
Fungal Genomic DNA Extraction and Multi-gene Sequencing
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Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified CTAB protocol as described in Guo et al. (2000) (link). Seven loci, including the 5.8S nuclear ribosomal RNA gene with the two flanking internal transcribed spacer (ITS) re gions, intergenic spacer region of the rDNA (IGS), partial trans lation elongation factor (tef1), partial calmodulin (cam), partial RNA polymerase largest subunit (rpb1), partial RNA polymerase second largest subunit (rpb2) gene regions, and partial β-tubulin (tub2), were amplified and sequenced, respectively. The primer pairs and PCR amplification procedures following protocols described by O’Donnell et al. (1998a , b (link), 2008 (link), 2009a (link), b (link), 2010 (link)), Crous et al. (2009 (link), 2021 (link)), and Lombard et al. (2015) (link), are listed in Table 2 . PCR amplifications were performed in a reaction mixture consisting of 12.5 μL 2 × Taq PCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 1 μL each of 10 μM primers, 1 μL of the undiluted genomic DNA, adjusted to a final volume of 25 μL with distilled deionized water. The PCR products were visualised on 1 % agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Tianyi Huiyuan Company (Beijing, China). Consensus sequences were obtained using SeqMan of the Lasergene software package v. 14.1 (DNAstar, Madison, Wisconsin, USA).
9
Fungal DNA Extraction and Sequencing
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Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified CTAB protocol as described in Guo et al. (2000) . Five loci, including the 5.8S nuclear ribosomal RNA gene with the two flanking internal transcribed spacer (ITS), translation elongation factor (EF-1α), calmodulin (CAM), partial RNA polymerase largest subunit (RPB1) and partial RNA polymerase second largest subunit (RPB2) gene regions, were amplified and sequenced, respectively. The primer pairs and PCR amplification procedures following protocols described by Crous et al. (2009) (link) are listed in Table 2 . PCR amplifications were performed in a reaction mixture consisting of 12.5 μL 2 × Taq PCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), 1 μL each of 10 μM primers, 1 μL of the undiluted genomic DNA, adjusted to a final volume of 25 μL with distilled deionized water. The PCR products were visualised on 1 % agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the TIANYI HUIYUAN Company (Beijing, China). Consensus sequences were obtained using SeqMan of the Lasergene soft-ware package v. 14.1 (DNAstar, Madison, Wisconsin, USA).
10
Validating Genetic Variants by PCR and Sanger Sequencing
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SNV and indels were validated by PCR reaction and Sanger sequencing. PCR primers were designed using the online software Primer3 (http://frodo.wi.mit.edu/primer3/ ) using GRCh37/hg19 as the reference sequence. We used a 96-well GeneAmp PCR System 9700 (Applied Biosystems) to perform the PCR reaction. A total of 10 ng of DNA from each sample was used per reaction with the 2× Taq PCR Master Mix (Vazyme Biotech). The sequencing reactions were performed with the Big Dye Terminator v.3.1 kit (Applied Biosystems). PCR products were sequenced using a 3730xl DNA Analyzer (Applied Biosystems).
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