A fragment of the ribosomal DNA of Trichobilharzia sp., spanning the sequences of internal transcribed spacers 1 and 2 (ITS1, ITS2), and 5.8S, 18S and 28S ribosomal RNA (rRNA) gene regions, was amplified using the specific primers its5Trem (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and its4Trem (5′-TCCTCCGCTTATTGATA TGC-3′) (12 (link)). The PCR was performed in 25μl reaction containing 4 μl of the genomic DNA, 2.5U of Taq DNA polymerase (Fermentas, Germany), 50 μM of each dNTPs (CinnaGen, Iran), 2 mM of MgCl2, 2.5μl of 10× PCR buffer and 0.5μM of each primer. The reaction was run in a Bioer XP thermal cycler (China) and comprised an initial DNA denaturation step at 95 °C for 5 min, followed by 35 cycles of DNA denaturation at 95°C for 60s, primer annealing at 50 °C for 45s, primer extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. A volume of 10 μl of each PCR product was analyzed by electrophoresis on 1% agarose gel at 120V for about 30 min. The gel was visualized by staining with ethidium bromide. The snail samples showing the band patterns corresponding to the gene regions of Trichobilharzia species were considered as infected.
Dntps
Manufactured by CinnaGen
DNTPs are a set of four distinct nucleotides that serve as the building blocks for DNA synthesis. These include dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (deoxythymidine triphosphate). DNTPs are essential components in various molecular biology techniques, such as PCR (Polymerase Chain Reaction) and DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by CinnaGen (5 mentions)
Mgcl2, by CinnaGen (4 mentions)
Gel imager, by Thermo Fisher Scientific (3 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (2 mentions)
Thermal cycler, by Bioer (1 mentions)
Accuprep gel purification kit, by Bioneer (1 mentions)
Mj mini thermal cycler, by Bio-Rad (1 mentions)
Cinnapure dna kit, by CinnaGen (1 mentions)
Pcr buffer, by CinnaGen (1 mentions)
Gel doc, by Uvitec (1 mentions)
Ethidium bromide, by CinnaGen (1 mentions)
100 bp dna ladder, by CinnaGen (1 mentions)
7 protocols using dntps
1
Molecular Identification of Trichobilharzia
2
Molecular Detection of dltS Gene
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Molecular detection was performed based on Poyart et al. (2007) (14 (link)), using the specific primer pairs of dlts-F and dlts-R (Table 1 ). Amplifications were done using 1X PCR buffer (10X, CinnaGen, Iran), 1.5 mM MgCl2 (50 mM, CinnaGen, Iran), 0.2 mM dNTPS (10 mM, CinnaGen, Iran), 10 pmol of each primer (dltS) (Takapouzist, Iran), and two units of Taq DNA polymerase (5 U/mL, CinnaGen, Iran). The PCR program was done by a first denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, and an extension at 72°C for 1 minute. The final extension was done at 72°C for 5 minutes. The amplicons were analyzed using 1% agarose gel and visualized with a gel imager (Life Technologies, USA).
3
Molecular Identification of Fasciola Species
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Genomic DNA was extracted from the Fasciola specimens by a modified phenol-chloroform method using cetyltrimethylammonium bromide (CTAB) (22 ). A fragment of the 28S ribosomal RNA (rRNA) gene of the Fasciola was amplified using two primers (forward: 5′-ACGTGATTACCCGCTGAACT-3′- and reverse: 5′-CTGAGAAAGTGCACT GACAAG-3′) (13 (link)). The PCR was carried out by 25μl reaction containing 2μl of the genomic DNA (diluted 1:30), 2.5U of Taq DNA polymerase (Fermentas, Germany), 50μM of each dNTPs (CinnaGen, Iran), 2mM MgCl2, 2.5μl PCR reaction buffer (10×) and 0.5μM of each primer. The reaction was performed in a Bioer XP thermal cycler and comprised an initial DNA denaturation step at 94 °C for 3min, followed by 30 cycles of DNA denaturation at 94 °C for 30s, primer annealing at 60 °C for 30s and primer extension at 72 °C for 60s, and finally, an extension step at 72 °C for 5min. A volume of 10 μl of each of the PCR products along with the positive (i.e. the PCR mixture including the known DNA samples of F. hepatica and F. gigantica) and negative (i.e. the PCR mixture excluding the DNA) controls were analysed by electrophoresis on 1.5% aga-rose gel for about 1.5h at 90V and visualized by staining with 1% ethidium bromide.
4
Multiplex PCR for Bacterial Serotyping
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Molecular serotyping was performed using two sets of multiplex PCR reactions (Table 1 ). Amplification of both multiplex reactions were done using 1X PCR buffer (10X, CinnaGen, Iran), 1.5 mM MgCl2 (50 mM, CinnaGen, Iran), 0.2 mM dNTPS (10 mM, CinnaGen, Iran), 10 pmol of each primer (Takapouzist, Iran), and two units of Taq DNA polymerase (5 U/mL, CinnaGen, Iran). The PCR program was carried out by the first denaturation at 94°C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 1 minute, annealing at 49.5°C and 60 °C for the first and second set, respectively, for 1 minute; and an extension at 72°C for 1minute. The final extension was done at 72 °C for 5 minutes (Applied Biosystems, ABI, Foster City, CA, USA). The amplicons were analyzed using 1% agarose gel and visualized with Gel imager (Life Technologies, USA).
5
Molecular Detection of Mycobacterium tuberculosis
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M. tuberculosis strain H37Rv was cultured on Lowenstein Jensen (LJ) medium and was incubated for six weeks at 37°C. DNA extraction was performed using boiling method (15 ).
The mpt64 primers were designed by Gene Runner software and were synthesized by CinnaGen Company of Iran. The mpt64 gene amplification was performed using the following primers, forward:
The DNA amplification involved initial denaturation at 95°C for 4 min, proceeded by 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 45 sec, followed by final extension at 72°C for seven minutes. Then, 100 μL of the PCR product was electrophoresed on 1% agarose gel and the mpt64 fragment purification was performed using AccuPrep® Gel Purification Kit (Bioneer, Korea).
The mpt64 primers were designed by Gene Runner software and were synthesized by CinnaGen Company of Iran. The mpt64 gene amplification was performed using the following primers, forward:
5′TATTTC
5′-CATATAT
The DNA amplification involved initial denaturation at 95°C for 4 min, proceeded by 35 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 45 sec, followed by final extension at 72°C for seven minutes. Then, 100 μL of the PCR product was electrophoresed on 1% agarose gel and the mpt64 fragment purification was performed using AccuPrep® Gel Purification Kit (Bioneer, Korea).
6
Multiplex PCR for Protein Subtyping
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Protein subtyping was performed using a multiplex PCR reaction (Table 3 ) (17 (link)). Amplification was done using 1X PCR buffer (10X, CinnaGen, Iran), 1.5 mM MgCl2 (50 mM, CinnaGen, Iran), 0.2 mM dNTPS (10 mM, CinnaGen, Iran), 10 pmol of each primer (Takapouzist, Iran), and two units of Taq DNA polymerase (5 U/mL, CinnaGen, Iran). The PCR program was done by first denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 35 seconds. The final extension was done at 72°C for 5 minutes. The amplicons were analyzed using 1.5% agarose gel and visualized with a gel imager (Life Technologies, USA).
7
RAPD-PCR Profiling of DNA Extracts
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DNA extraction was done using the CinnaPure DNA kit (CinnaGen, Iran) as described by the manufacturer. RAPD-PCR reactions were performed with oligonucleotide primers OLP6 (5′-GAGGGAAGAG-3′), OLP11 (5′-ACGATGAGCC-3′) and OLP13 (5′-ACCGCCTGCT-3′) as described by Williams et al. (1990) (link). Amplifications were carried out in a total volume of 25 μl containing 0.75 μl of oligonucleotides primers (3 μM), 0.75 μl dNTPs (200 μM of each deoxynucleoside triphosphate) (CinnaGen, Iran), 1.5 μl of MgCl2 (3.5 mM) (CinnaGen, Iran), 0.2 μl of Taq DNA polymerase (2.5 U) (CinnaGen, Iran), 2.5 μl of 10X PCR buffer (CinnaGen, Iran), 16.5 μl of sterile distilled water. Rather than “3μl” an estimate of the amount of DNA (ng) is needed.
Amplification of DNA fragments was performed in a MJ Mini Thermal Cycler (BIO-RAD, USA) with initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 93 °C for 1 min, annealing at 37 °C for 90 S and extension at 72 °C for 1 min, with a final extension of 7 min at 72 °C.
Amplified products were then resolved by electrophoresis in 1.5% agarose gel (CinnaGen, Iran) containing 0.5 μg/ml ethidium bromide (CinnaGen, Iran) and visualized under UV light by Gel Doc (UVitec, UK). A 100 bp DNA ladder (CinnaGen, Iran) was used as a DNA fragment size marker in all gels.
Amplification of DNA fragments was performed in a MJ Mini Thermal Cycler (BIO-RAD, USA) with initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 93 °C for 1 min, annealing at 37 °C for 90 S and extension at 72 °C for 1 min, with a final extension of 7 min at 72 °C.
Amplified products were then resolved by electrophoresis in 1.5% agarose gel (CinnaGen, Iran) containing 0.5 μg/ml ethidium bromide (CinnaGen, Iran) and visualized under UV light by Gel Doc (UVitec, UK). A 100 bp DNA ladder (CinnaGen, Iran) was used as a DNA fragment size marker in all gels.
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