Conventional PCR was done using Taq DNA polymerase 2x Master Mix RED which is a 2x reaction mix with the Ampliqon Taq DNA polymerase, NH4+ buffer system and magnesium chloride present. Twenty five μl of the 2x Master Mix RED was used for each reaction. The primer sequence used was Forward: 5'-AGG CAG CTT GCC ATA CTG CG-3' Reverse: 5'-ACT GTT AGC AAC TAC CGA TGT-3'. The agarose mix was prepared in 1x–TAE buffer in a microwave oven to get a clear homogeneous solution. The solution was cooled, and ethidium bromide was added. Then, the solution was poured in to the gel mold with the combs placed. After allowing it to settle for 30 min, the comb was removed carefully, and tapes were peeled off from the gel casting tray. The gel was kept completely submerged in the buffer. Each of the PCR products with a quantity of 10 μl was taken and mixed with gel loading dye and then loaded to ethidium bromide incorporated in standard 2% agarose gel (0.5 μg/ml). A DNA marker (100 base pair ladder) was at the same time loaded in a separate lane. The gel was run at 80V for 30 min or until dye reaches two-third of the gel. PCR products in the gel were viewed on an ultraviolet transilluminator (BIORAD) [Figures 5 and 6 ].
Master mix red
Manufactured by Ampliqon
Sourced in Denmark
Master Mix RED is a ready-to-use polymerase mix designed for efficient and robust PCR amplification. It contains all the necessary components, including a DNA polymerase, PCR buffer, and dNTPs, to perform PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by Ampliqon (2 mentions)
High pure pcr template preparation kit, by Roche (2 mentions)
Thermal cycler, by Thermo Fisher Scientific (1 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Genomic dna isolation kit, by GeNet Bio (1 mentions)
Peqstar, by Avantor (1 mentions)
Mspi restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Sabouraud dextrose agar, by Merck Group (1 mentions)
Thermal cycler, by Bio-Rad (1 mentions)
Gelred nucleic acid gel stain, by Biotium (1 mentions)
Pcr master mix, by Ampliqon (1 mentions)
Mastercycler ep gradient s thermocycler, by Eppendorf (1 mentions)
24 protocols using master mix red
1
Conventional PCR Using Taq Polymerase
2
Biocide Resistance Genes Detection
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The presence of qacE, qacE∆1, and sugE1 genes that confer resistance to biocides was examined using the primers shown in Table 6 . PCRs were conducted on a thermal cycler (Applied Biosystems, USA) in 25 μl reaction volume containing 10 μL of 2X Master Mix RED (Ampliqon, Denmark), 1 μl of 10 pmol of each primer (Sinaclon Co; Tehran, Iran), 50 ng of template DNA and 6 μl of sterile distilled water. PCR conditions were performed under the following thermal conditions: pre-denaturation at 94 °C for 5 min; 30 cycles of DNA denaturation for 1 min at 94 °C; annealing at 51–54 °C, according to the primers for each gene (Table 6 ) for 25 s, extension for 50 s at 72 °C and a final extension at 72 °C for 7 min. All of the amplified products were separated by electrophoresis in 1.8% agarose gel stained with green viewer (Parstous, Mashhad. Iran). PCR experiment was run in triplicate (from the same sample) for all isolates tested. A representative PCR amplicon of each gene with the corresponding PCR primers was sequenced by Sanger technology to ensure the specific amplification. The sequenced data obtained was viewed in Chromas software and alignment were conducted using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ).
3
Amplification and Sequencing of Eg14-3-3 Gene
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Eg14-3-3 gene was amplified by polymerase chain reaction (PCR). The PCR reaction performed in a 25 µL reaction volume as follows: 9.5 µL DNase free deionized water, 1 µL template DNA (1017.6 ng/µL), 1 µL each primer (100 pmoles/µL) and 12.5 µL PCR master mix (2x Master Mix RED, Taq DNA polymerase, 0.5 µM of dNTPs and 1.5 mM MgCl2) obtained from Ampliqon (Odense, Denmark). Based on NCBI’s Eg14-3-3 gene sequence (GenBank accession No: AY942149), the forward and reverse primers were designed using OLIGO 7 software.16 (link) The primers were as follows: the forward primer, 5'-ATGTCTTCTCTCAGTAAGCGCGA-3' and the reverse primer, 5'-ATCGGCTTTCGGCGGTTCAG-3'. The gene amplification was conducted using following condition: (i) one cycle at 94°C for 4 minutes as primary denaturation, (ii) 32 cycles at 94 °C for 1 minute (denaturation), 52°C for 1 minute (annealing) and 72°Cfor 1 minute (extension), and (iii) three cycles as final extension (72°C for 10 minutes). The PCR products were visualized by electrophoresis on a 1% agarose gel and size of the product analyzed with 1 kb DNA ladder (GeneRulerTM 1 kb Plus DNA ladder), obtained from Fermentase (Thermo Fisher Scientific, Waltham, USA). The PCR-amplified product was sequenced, and its mRNA sequence was registered in NCBI GenBank as Iran Eg14-3-3 isolate (GenBank: KU739136).
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Eg14-3-3 gene was amplified by polymerase chain reaction (PCR). The PCR reaction performed in a 25 µL reaction volume as follows: 9.5 µL DNase free deionized water, 1 µL template DNA (1017.6 ng/µL), 1 µL each primer (100 pmoles/µL) and 12.5 µL PCR master mix (2x Master Mix RED, Taq DNA polymerase, 0.5 µM of dNTPs and 1.5 mM MgCl2) obtained from Ampliqon (Odense, Denmark). Based on NCBI’s Eg14-3-3 gene sequence (GenBank accession No: AY942149), the forward and reverse primers were designed using OLIGO 7 software.16 (link) The primers were as follows: the forward primer, 5'-ATGTCTTCTCTCAGTAAGCGCGA-3' and the reverse primer, 5'-ATCGGCTTTCGGCGGTTCAG-3'. The gene amplification was conducted using following condition: (i) one cycle at 94°C for 4 minutes as primary denaturation, (ii) 32 cycles at 94 °C for 1 minute (denaturation), 52°C for 1 minute (annealing) and 72°Cfor 1 minute (extension), and (iii) three cycles as final extension (72°C for 10 minutes). The PCR products were visualized by electrophoresis on a 1% agarose gel and size of the product analyzed with 1 kb DNA ladder (GeneRulerTM 1 kb Plus DNA ladder), obtained from Fermentase (Thermo Fisher Scientific, Waltham, USA). The PCR-amplified product was sequenced, and its mRNA sequence was registered in NCBI GenBank as Iran Eg14-3-3 isolate (GenBank: KU739136).
4
Molecular Detection of AdeABC System in Acinetobacter baumannii
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The standard PCR assay was performed using specific primers for detection of structural adeB gene and two regulatory genes, adeR and adeS belong to AdeABC system in tetracycline-resistant A. baumannii isolates the DNA amplification instrument Mastercycler gradient to detect one structural (adeB) and two regulatory (adeR and adeS) genes of AdeABC. The genomic DNAs for PCR were prepared by boiling method. Three pairs of oligonucleotide primers used for the reactions are listed in Table 1 . The PCR was performed in a reaction mixture with total volume of 25 μl containing 2x Master Mix Red (Ampliqon, Denmark) (containing 10 pmolTaq DNA Polymerase), 0.5 mM of each primer and 1 μl of DNA suspension. The PCR program was performed as follows: Initial denaturation at 94°C for 5 minutes; 30 cycles containing of denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute, and extension at 72°C for 1 minute; followed by a final extension at 72°C for 5 minute. The amplified products were subjected to electrophoresis in 1% agarose gels.
5
Multiplex PCR Detection of Staphylococcus Genes
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Amplification was conducted in temperature gradient thermal cycler (Biometra-T300, Gottingen, Germany) in a volume of 25µl. Each 25µl PCR mixture consisted of 1µl of bacterial DNA, 0.5 µl (10pM) of each oligodeoxynucleotide primers, 12.5 µl of 2× Master Mix Red (Ampliqon, Denmark) and 11µl DNase and RNase free water. PCR was used for detection nuc, mecA, icaA, icaD, icaB,icaC,scn, sea, sak, sep, chb genes. All primers and programs can be found in Table I [14 (link), 18 (link), 26 (link), 28-30 (link)]. After amplification, the PCR products were analyzed by electrophoresis on 1.5% agarose gel in 0.5×TBE buffer (5.4 g Tris base, 2.75 g boric acid, 2 ml 0.5 M EDTA, in 1 L). DNA ladder was a ready to use plasmid double digest sized range 100-3000bp obtained from SMOBIO Technology (Hsinchu, Taiwan). Specificity of the primers was checked by Primer Quest software tool (http://www.ncbi.nlm.nih.gov/GenBank ).
6
Genotyping rs2069740 and rs34255686 Polymorphisms
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After collecting 5 mL peripheral blood samples in EDTA tubes, genomic DNA was extracted using a Genomic DNA Isolation Kit (GeNet Bio, Daejeon, Korea), using the salting out method. To genotype rs2069740(T/A) and rs34255686(C/A) polymorphisms, the amplification refractory mutation system PCR (ARMS-PCR) method was carried out. For each polymorphism, two external primers (to check the performance of the PCR) and two allele-specific internal primers (to detect each allele) were designed (the designed primer sequences are reported in Table 2 ). PCR was performed in a total volume of 23.1 μL, including 1 μL of template DNA, 11 μL of 2× Master Mix RED (Ampliqon), 0.8 μL (0.39 pmol) of each primer and 7.9 μL of H2O. The cycling conditions were as follows: initial denaturation for 5 min at 94 °C, followed by 30 cycles at 94 °C for 40 s (denaturation), annealing temperature as in Table 2 for 40 s, 1 min at 72 °C (extension) and a final extension for 7 min at 72 °C. PCR products were run by standard electrophoresis on 2% agarose gel for 10 min and visualized on a UV transilluminator. The band lengths are presented in Table 2 .
7
Genomic DNA Extraction and Parasite Detection
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Genomic DNA of the samples was extracted using the following protocol. Briefly, 50 mg of each sample were frozen and thawed for three times (each for 10 min). DNA was extracted using High Pure PCR Template Preparation Kit (Roche, Germany) according to the manufacturer's instructions. Extracted DNA was stored at -20 °C for PCR amplification. The extracted DNA was used as template to detect eucoccidial parasites using conventional polymerase chain reaction (PCR). Fragments of nearly 900-bp from 18S ribosomal DNA genes were amplified using single PCR and forward (SarcoFext 5′-GGTGATTCATAGTAACCCAACG-3′) and reverse (SarcoRext 5′-GATTTCTCATAAGGTGCAGGAG-3′) primers (More et al., 2014 (link)). The PCR amplification was carried out with 20-μl reaction volumes, including 10 μl of 2× Master Mix RED (Ampliqon, Denmark) with 1.5 mM of MgCl2, 1 μl of each primer at concentration of 10 pmol, 6.5 μl of sterile distilled water and 1.5 μl of the template DNA. The PCR reactions were amplified using thermal cycler (Peqlab peqSTAR, USA) with the following cycling conditions: initial hot start at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 45 s. Final extension was carried out at 72 °C for 5 min. The PCR products were analyzed using electrophoresis on 1% agarose gel and UV visualization.
8
DNA Extraction and PCR Amplification Protocol
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Genomic DNA was extracted using boiling method as previously described (16 (link)). All PCR reactions were carried out by a Gradient thermal cycler (Biometra-T300, Gottingen, Germany) in a final reaction mixture volume of 25 μl containing 1 μl of genomic DNA, 0.5 μl (10 pM) of each oligodeoxynucleotide primers, 12.5 μl of 2× Master Mix Red (Ampliqon, Co, Denmark) and 11 μl DNase and RNase free water. After amplification, the PCR products were electrophoresed on 1.5% agarose gel electrophoresis in TBE 0.5× buffer (5.4 g Tris base, 2.75 g Boric acid, 2 ml 0.5 M EDTA, in 1 L) at 100 V for 90 min. The products were detected by staining with Green Viewer Dye and then photographed. The oligonucleotide primers as well as PCR programs are presented in Table 1 .
9
Molecular Identification of Fungal Isolates
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DNAs that were extracted from a 24-hour fresh colony, were cultured on Sabouraud Dextrose agar at 37°C (Merck, Germany) according to the isopropanol and the proteinase K method.18 (link)
The PCR reaction mixture was prepared as follows: 1 μL of DNA, 10 µl of Master Mix RED (Ampliqon, Denmark), and 0.5 µl of each ITS1/ ITS4 primer in a total volume of 25 μL. The PCR program conditions were 95 °C for 10 minutes, 35 cycles of denaturation for 40 seconds at 95 °C, annealing for 40 seconds at 56 °C, an extension for 40 seconds at 72 °C, with the last extension of 8 minutes at 72 °C. The amplicons were analyzed by 1.5% agarose gel electrophoresis in Tris base boric acid EDTA (TBE) buffer (Merck, Germany) using the DNA Safe Stain (Fermentas, USA). The 10 microliters of products were subjected to the MSP I restriction enzyme (Fermentas, USA). Restriction fragments were separated by 2% agarose gel electrophoresis.
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DNAs that were extracted from a 24-hour fresh colony, were cultured on Sabouraud Dextrose agar at 37°C (Merck, Germany) according to the isopropanol and the proteinase K method.18 (link)
The PCR reaction mixture was prepared as follows: 1 μL of DNA, 10 µl of Master Mix RED (Ampliqon, Denmark), and 0.5 µl of each ITS1/ ITS4 primer in a total volume of 25 μL. The PCR program conditions were 95 °C for 10 minutes, 35 cycles of denaturation for 40 seconds at 95 °C, annealing for 40 seconds at 56 °C, an extension for 40 seconds at 72 °C, with the last extension of 8 minutes at 72 °C. The amplicons were analyzed by 1.5% agarose gel electrophoresis in Tris base boric acid EDTA (TBE) buffer (Merck, Germany) using the DNA Safe Stain (Fermentas, USA). The 10 microliters of products were subjected to the MSP I restriction enzyme (Fermentas, USA). Restriction fragments were separated by 2% agarose gel electrophoresis.
10
Multiplex PCR Assay for Virulence Genes
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In this study, 11 different virulence gene factors (fimA, papC, papGIII, aer, sat, afa, sfa, cnf-1, hly, iutA, and iroN) were assessed by PCR method using a thermal cycler (Bio-Rad Laboratories, Inc.). The multiplex PCR reaction was performed in 25 μL volumes containing 3 μl of DNA template, 1 μl of each specific primer, 12.5 μL of Master Mix Red (Ampliqon, Denmark), and 7.5 μl of double-distilled water. The sequences of used primers and amplification conditions are presented in Table 1 [25 (link)–27 (link)]. The PCR products were analyzed by 1.8% agarose gel electrophoresis in 1x TBE buffer (0.89 M Tris-Borate, 0.02 M EDTA, pH 8.3), stained with ethidium bromide (SinaClon BioScience Co., Iran), and visualized by using UV light.
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