DNA was extracted from one single colony for each isolate in a final volume of 100 μL of distilled water by incubation at 95 °C for 10 min followed by a centrifugation step. The presence of blaCTX-M (CTX-M group 1, 2, 8, 9 and 25), blaTEM, blaSHV and blaOXA-like genes was assessed by multiplex PCR according to a previously published method [9 (link)]. DNA from reference blaCTX-M, blaTEM, blaSHV and blaOXA-like-positive strains was used as positive control. PCR products were visualized after electrophoresis on 1.5 % agarose gels containing ethidium bromide at 100 V for 80 min. A 100 bp DNA ladder (Promega, USA) was used as a marker size. PCR products were purified using the ExoSAP-IT purification kit (GE Healthcare, Piscataway, NJ, USA) and sequenced bidirectionally on a 3100 ABI Prism Genetic Analyzer (Applied Biosystems). Nucleotide sequence alignment and analyses were performed online using the BLAST program available at the National Center for Biotechnology Information web page http:// www.ncbi.nlm.nih.gov .
Exosap it purification kit
Manufactured by GE Healthcare
Sourced in United States
The ExoSAP-IT purification kit is a laboratory reagent used to remove excess primers and nucleotides from DNA samples prior to downstream applications such as sequencing or PCR analysis. The kit utilizes enzymatic treatment to efficiently purify the DNA.
Automatically generated - may contain errors
Lab products found in correlation
Taq polymerase 5 u, by Promega (2 mentions)
Bigdye terminator kit version 1, by Thermo Fisher Scientific (2 mentions)
100 bp dna ladder, by Promega (1 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Sequence scanner, by Thermo Fisher Scientific (1 mentions)
3730xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator kit, by Thermo Fisher Scientific (1 mentions)
Mgcl2, by Promega (1 mentions)
4 protocols using exosap it purification kit
1
Multiplex PCR for Detecting Antibiotic Resistance Genes
2
16S rRNA Gene Amplification and Sequencing
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Total DNA extracted from samples was used as a template for the amplification of a fragment of the 16S ribosomal DNA by polymerase chain reaction (PCR). Amplification was performed using the highly conserved universal primers: Fd1 5′-AGAGTTTGATCCTGGCTCAG-3′ and RP2 5′-ACGGCTACCTTGTTACGACTT-3′ [25 ].
The PCR reactions were carried out using a TC1000-G thermocycler (DLAB Scientific), in a total volume of 15 μL containing approximately 100 ng of genomic DNA, 0.3 μL of each 10 mm dNTP (Promega), 3 μl of 5X buffer (Promega), 0.9 μL of 25 mm MgCl2 (Promega), 0.12 μL of each 10 mm primer, and 0.075 μL of Taq polymerase 5 U (Promega). The PCR was programmed as follows: 95°C, 2′/(95°C, 40″-55°C, 40″-72°C, 1′) ×35; 72°C/5′/4°C.
The PCR products were purified using the ExoSAP-IT purification kit (GE Healthcare), and sequencing was performed with the BigDye Terminator Kit version 1.0 (Applied Biosystems).
Sequencing products were separated and detected in a 3730xl Genetic Analyzer (Applied Biosystems). Chromatogram revision and trimming was performed with the sequence scanner (Applied Biosystems), and alignment was obtained from ClustalX implemented in BioEdit [26 ].
The PCR reactions were carried out using a TC1000-G thermocycler (DLAB Scientific), in a total volume of 15 μL containing approximately 100 ng of genomic DNA, 0.3 μL of each 10 mm dNTP (Promega), 3 μl of 5X buffer (Promega), 0.9 μL of 25 mm MgCl2 (Promega), 0.12 μL of each 10 mm primer, and 0.075 μL of Taq polymerase 5 U (Promega). The PCR was programmed as follows: 95°C, 2′/(95°C, 40″-55°C, 40″-72°C, 1′) ×35; 72°C/5′/4°C.
The PCR products were purified using the ExoSAP-IT purification kit (GE Healthcare), and sequencing was performed with the BigDye Terminator Kit version 1.0 (Applied Biosystems).
Sequencing products were separated and detected in a 3730xl Genetic Analyzer (Applied Biosystems). Chromatogram revision and trimming was performed with the sequence scanner (Applied Biosystems), and alignment was obtained from ClustalX implemented in BioEdit [26 ].
3
PCR Purification and Sequencing
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ExoSAP-IT purification kit from G.E. Healthcare was used to purify the PCR products before proceeding to the sequencing. The sequencing was carried out using the BigDye Terminator Kit version 1.0 (Applied Biosystems). The obtained isolate's gene sequence was examined using BLAST implemented in Geneious Prime Software (2022.2). BioEdit 7.2 was used to estimate the sequence identity values.
4
Detailed 16S rRNA Sequencing Protocol
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A chloroform phenol technique was used to extract DNA, as previously described (Derdak et al., 2021 (link)). Total genomic DNA was isolated from samples and utilized as a template for polymerase chain reaction amplification of a 16S ribosomal DNA fragment (PCR). The PCR reactions have been conducted through a DLAB TC1000-G thermocycler. Two pairs of conserved universal primers were used for amplification: Fd1 5′-AGAGTTTGATCCTGGCTCAG-3 'and RP2 5′-ACGGCTACCTTGTTACGACTT-3′.
The PCR was performed in a total volume of 15 µL composed of approximately 100 ng of genomic DNA, 0.3 μL of each 10 mM dNTP (Promega), 3 μL of 5X buffer (Promega), 0.9 μL of 25 mM MgCl2 (Promega), 0.12 μL of each 10 mM primer and 0.075 µL of Taq polymerase 5U (Promega). The PCR program used was as follow:
95 °C, 2 '/(95 °C, 40′ '- 55 °C, 40′ '- 72 °C, 1′) x35; 72 °C/5 '/4 °C.
The ExoSAP-IT purification kit (GE Healthcare) was used to purify the PCR products, and the BigDye Terminator Kit version 1.0 was used to sequence them (Applied Biosystems). The sequences obtained were corrected using BioEdit, introduced into Geneious prime and aligned via BLAST to determine the degree of similarity of our sequences with those present in the database.
The PCR was performed in a total volume of 15 µL composed of approximately 100 ng of genomic DNA, 0.3 μL of each 10 mM dNTP (Promega), 3 μL of 5X buffer (Promega), 0.9 μL of 25 mM MgCl2 (Promega), 0.12 μL of each 10 mM primer and 0.075 µL of Taq polymerase 5U (Promega). The PCR program used was as follow:
95 °C, 2 '/(95 °C, 40′ '- 55 °C, 40′ '- 72 °C, 1′) x35; 72 °C/5 '/4 °C.
The ExoSAP-IT purification kit (GE Healthcare) was used to purify the PCR products, and the BigDye Terminator Kit version 1.0 was used to sequence them (Applied Biosystems). The sequences obtained were corrected using BioEdit, introduced into Geneious prime and aligned via BLAST to determine the degree of similarity of our sequences with those present in the database.
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