In the bacterial isolates containing the IntI1 and the IntI2 genes, the variable regions (VRs) of both of the studied integrons were sequenced in order to reveal the specific DNA sequence (plausibly bearing the multidrug resistance genes) within the integron structure. The regions were amplified with a set of specific primers: Integron CL (for integron 1) and Integron CL JJ (for integron 2), sequences are listed in Table 6 . The PCR reaction conditions, and primer concentrations were the same as described for IntI1 and IntI2 amplification, with an annealing temperature of 61 °C. The amplified gene cassettes of similar PCR product length (base pair, bp) were sequenced by the Sanger method. Prior to sequencing, the PCR products were purified with FastAP and ExoI enzymes (EF0654 and EN0581, Thermo Fisher Scientific, Waltham, Massachusetts, USA), amplified With the BigDye™ terminator v3.1 Cycle Sequencing Kit (4,337,458, Life Technologies, Carlsbad, California, USA) and purified on Sephadex G50 (G5050, Sigma, St. Louis, Missouri, USA) by filtration. Sequencing of the cassette arrays was done with Applied Biosystems ABI 3130xl 16-capillary array genetic analyzer (Applied Biosystems, Waltham, Massachusetts, USA). Data was analysed with the Seqman software.
Exoi enzymes
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
ExoI enzymes are a class of exonucleases that catalyze the stepwise removal of mononucleotides from the 3' end of single-stranded DNA or RNA molecules. These enzymes are used in various molecular biology applications, such as DNA sequencing and DNA repair analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sephadex g 50, by Merck Group (1 mentions)
Fastap, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Bigdye v3.1 sequencing buffer, by Thermo Fisher Scientific (1 mentions)
Abi prism 3500xl genetic analyser, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator mix v3, by Thermo Fisher Scientific (1 mentions)
2 protocols using exoi enzymes
1
Integron Sequencing for Multidrug Resistance
2
Amplicon Purification and Sanger Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Amplicons were purified for sequencing by adding 2μl FastAP (1U/ μl) and 0.5μl (20U/ μl) ExoI enzymes (Thermo, Vilnius, Lithuania) to 19μl of the PCR product and incubated at 37°C for 15 min. This was followed by an 85°C for 15 min incubation.
Sanger sequencing reactions on 2μl of the purified PCR was by adding 1µl BigDye ® Terminator mix v3.1 (Applied Biosystems, Foster City, CA, USA), 2.25µl 5x BigDye ® v3.1 sequencing buffer, 0.75µl Univ-p33-F primer (2µM) and molecular grade water to a total volume of 10µl and using 1 cycle of 94°C for 1 min, 30 cycles of 94°C for 10
RNA isolation, reverse transcription and PCR amplification seconds, 50°C for 5 seconds and 60°C for 4 minutes. Sequencing products were purified using ethanol precipitation, according to Sambrook (2001) . The purified sequencing products were submitted to the African Centre for Gene Technologies (ACGT), Automated Sequencing Facility, Department of Genetics, University of Pretoria, South Africa and sequenced using an ABI Prism® 3500xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequences not conforming to a quality criterion of a minimum PHRED score of 30 were discarded from further analysis.
Sanger sequencing reactions on 2μl of the purified PCR was by adding 1µl BigDye ® Terminator mix v3.1 (Applied Biosystems, Foster City, CA, USA), 2.25µl 5x BigDye ® v3.1 sequencing buffer, 0.75µl Univ-p33-F primer (2µM) and molecular grade water to a total volume of 10µl and using 1 cycle of 94°C for 1 min, 30 cycles of 94°C for 10
RNA isolation, reverse transcription and PCR amplification seconds, 50°C for 5 seconds and 60°C for 4 minutes. Sequencing products were purified using ethanol precipitation, according to Sambrook (2001) . The purified sequencing products were submitted to the African Centre for Gene Technologies (ACGT), Automated Sequencing Facility, Department of Genetics, University of Pretoria, South Africa and sequenced using an ABI Prism® 3500xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Sequences not conforming to a quality criterion of a minimum PHRED score of 30 were discarded from further analysis.
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