Determination of phylogenetic origin was carried out following the revised method described by Clermont et al. [19 (link)]. The method is based on a quadruplex PCR reaction to detect the genes arpA, chuA, yjaA, and TspE4.C2. The pattern of amplification for those four markers in that order defines the phylogroups as follows: A (+ − − -), B1 (+ − − +), B2 (− + + +) or (− + + −) or (− + − +), and F (− + − −). Strains presenting other amplification patterns were submitted to a second PCR reaction for the distinction between phylogroups A and C, D and E, or E and clade I according to Clermont et al. [19 (link)]. Prototype strains E. coli RS218, and EDL933 were used as positive controls. Reactions were performed in a Mastercycle Gradient Thermocycler (Mastercycle gradient – Eppendorf) using boiled isolated colonies as template DNA, primers from IDT (Integrated DNA Technologies - USA), and Go Taq Green Master Mix (Promega - USA). The resulted amplified DNA fragments were resolved on 2% agarose gels, stained and photographed in a digital image capture system (Universal Hood II – Biorad Laboratories, Inc. USA). “1Kb Plus DNA Ladder” (Invitrogen™ - Thermo Fisher Scientific) was used as a mass molecular marker.
Mastercycle gradient thermocycler
Manufactured by Eppendorf
Sourced in United States, Germany
The Mastercycle Gradient Thermocycler is a laboratory instrument designed for DNA amplification by polymerase chain reaction (PCR). It provides precise temperature control and gradient capabilities to optimize PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Universal hood 2, by Bio-Rad (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Bacterial genomic dna extraction kit, by Takara Bio (1 mentions)
Spss version 18.0 for windows, by IBM (1 mentions)
Gelred, by Biotium (1 mentions)
2 protocols using mastercycle gradient thermocycler
1
Phylogenetic Typing of E. coli Strains
2
Multiplex PCR for Virulence, Resistance, and Biofilm
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M-PCRs reactions were performed for detection of virulence, resistance and bio lm corresponding genes.
Chromosomal DNA was extracted from the pure colonies using the Bacterial Genomic DNA Extraction kit (TaKaRa Biotechnology Co., Ltd, Dalien, China). The DNA concentration and purity were evaluated using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scienti c, UK) and then kept at -20 °C until further use. The details of the primers used in this study are shown in Table 1. The process of M-PCR reactions in the nal volume of 25 μl was performed according to Table 2 in an Eppendorf MasterCycle Gradient Thermocycler (Eppendorf, Hamburg, Germany). M-PCRs products were electrophoresed in a 1% agarose/0.5 × TBE (45 mM-Tris-borate, 1 mM-EDTA) gel stained with 0.1 μl/ml Gel Red™ (Biotium, USA), then photographed under an UV trans-illuminator (Tanon, China).
Data analysis SPSS version 18.0 for Windows (SPSS Inc., Chicago, USA) was used for statistical analysis. P ≤ 0.05 was considered as a statistical signi cance.
Chromosomal DNA was extracted from the pure colonies using the Bacterial Genomic DNA Extraction kit (TaKaRa Biotechnology Co., Ltd, Dalien, China). The DNA concentration and purity were evaluated using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scienti c, UK) and then kept at -20 °C until further use. The details of the primers used in this study are shown in Table 1. The process of M-PCR reactions in the nal volume of 25 μl was performed according to Table 2 in an Eppendorf MasterCycle Gradient Thermocycler (Eppendorf, Hamburg, Germany). M-PCRs products were electrophoresed in a 1% agarose/0.5 × TBE (45 mM-Tris-borate, 1 mM-EDTA) gel stained with 0.1 μl/ml Gel Red™ (Biotium, USA), then photographed under an UV trans-illuminator (Tanon, China).
Data analysis SPSS version 18.0 for Windows (SPSS Inc., Chicago, USA) was used for statistical analysis. P ≤ 0.05 was considered as a statistical signi cance.
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