DNA was extracted from pure colonies of C. perfringens that showed a double zone of haemolysis on blood agar by using an extraction kit (QIA amp Mini Kit, Qiagen, Germany). Specific oligonucleotide primers for the alpha (cpa), beta (cpb), epsilon (etx), iota (ia), and enterotoxin toxin (cpe) genes of C. perfringens were used as described in Table 1 . Multiplex PCR assay was carried out in a reaction mixture (25 uL) containing 1 uL of template DNA, 12.5 uL of Dream Taq Green PCR Master mix, 0.5 uL of each primer (10 pmol/uL), and 10.5 uL of DNase-free water. PCR amplification was carried out in a Biometra PCR thermal cycler. Following initial denaturation for 3 min at 94°C, the samples were subjected to 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and final extension for 10 min at 72°C (28 (link)). The PCR reaction mixtures were analysed by electrophoresis using 1.5% (w/v) agarose gel in the presence of 100 bp DNA ladder (Fermentas Life Science, EU) (14 ). Types D and E, and positive-control of national C. perfringens strains were also included in the PCR.
Pcr thermal cycler
Manufactured by Analytik Jena
Sourced in Germany
The PCR thermal cycler is a laboratory instrument that is used to amplify DNA sequences through the process of Polymerase Chain Reaction (PCR). It precisely controls the temperature and duration of the various steps involved in the PCR process, including denaturation, annealing, and extension, to facilitate the exponential replication of target DNA fragments.
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Lab products found in correlation
4 protocols using pcr thermal cycler
1
Multiplex PCR for Clostridium perfringens
2
Quantitative Analysis of EZH2 Splicing
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RT-PCR reaction was accomplished as described previously (Wang et al., 2017 (link)). Briefly, 1 μg of total RNA was used for reverse transcription with prime Script Reverse Transcriptase (TAKARA, Japan). The produced cDNA was used for PCR reaction by using the following primers and PCR cycles. Cycle conditions were as follows: 94°C for 2 min; followed by 33 cycles of 94°C denaturation for 10 s, 58°C annealing for 15 s, and 72°C elongation for 30 s; with a final incubation at 72°C for 2 min in a PCR Thermal Cycler (BIOMETRA). PCR products were separated by electrophoresis and stained with ethidium bromide. The primers for PCR are as follows; EZH2 exon9 F, 5′-AAGCGGAAGAACACAGAAAC-3′, EZH2 exon10 R, 5′-CAGAGGAGCTCGAAGTTTCA-3′, For quantitation analysis of alternative splicing products, the signals were measured by ImageJ software [U.S. National Institutes of Health, Bethesda (Schneider et al., 2012 (link))].
3
Comprehensive Molecular Profiling Techniques
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The following were used in our experiments: polymerase chain reaction (PCR) thermal cycler (Biometra, Göttingen, Germany); stereoscopic microscope (Motic, Xiamen, China); proteinase K, deoxynucleotides (Shanghai Univ-bio Company, Shanghai, China); PCR primers (Invitrogen Trading Shanghai Co., Ltd., Shanghai, China) ; Taq polymerase, Tris-saturated phenol, chloroform (Sigma Co., Ltd., St Louis, MO, USA); rabbit polyclonal anti-SP-C and anti-Aqp5 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-β-actin, anti-Snx5, and anti-T1α antibodies (Abcam, Cambridge, MA, USA).
4
Cloning and Sequencing of Bull SPAM1 cDNA
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Total cellular RNA was extracted from the bull testis using ISOGEN (Nippon Gene, Japan) and cDNA was synthesized by oligo (dT) priming using the SuperScript III First-Strand Synthesis System (Invitrogen, USA) [2] . To amplify the cDNA fragments encoding bull SPAM1, two oligonucleotide primers were designed based on a bovine expressed sequence tag (GenBank Accession No. NM_001008413.3). The oligonucleotide sequences for the primers were as follows: 5'-CTCGAGGCCACCATGAGAATG CTGAGGCGCCACCA-3' (sense) and 5'-CTCGAGTTAATAGGT TGTTTGATTTTTAAT-3' (antisense).
Polymerase chain reaction (PCR) was performed for 35 cycles at 94°C for 60 sec, 60°C for 60 sec, and 72°C for 90 sec with a PCR thermal cycler (Biometra, Germany). The reverse transcription-PCR products corresponding to the bSPAM1 gene were cloned and sequenced. Following electrophoresis on a 1% agarose gel, the desired PCR band was excised with a razor blade. The gel fragment was purified using a gel extraction kit (Elpis Biothech, Korea) in accordance with the manufacturer's guidelines. The purified DNA fragment was then cloned in Escherichia coli DH5α cells using the pEGM-T easy kit (Promega, USA). After isolation of plasmid DNA, inserts were sequenced with the vector-specific T7 and SP6 sequencing primers (Promega, USA) on an ABI 3100 DNA sequencer (Applied Biosystems, USA).
Polymerase chain reaction (PCR) was performed for 35 cycles at 94°C for 60 sec, 60°C for 60 sec, and 72°C for 90 sec with a PCR thermal cycler (Biometra, Germany). The reverse transcription-PCR products corresponding to the bSPAM1 gene were cloned and sequenced. Following electrophoresis on a 1% agarose gel, the desired PCR band was excised with a razor blade. The gel fragment was purified using a gel extraction kit (Elpis Biothech, Korea) in accordance with the manufacturer's guidelines. The purified DNA fragment was then cloned in Escherichia coli DH5α cells using the pEGM-T easy kit (Promega, USA). After isolation of plasmid DNA, inserts were sequenced with the vector-specific T7 and SP6 sequencing primers (Promega, USA) on an ABI 3100 DNA sequencer (Applied Biosystems, USA).
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