Blood samples (1 mL) were collected, and genomic DNA was extracted from white blood cells using the TIANamp Blood DNA Kit [Tiangen Biotect (Beijing) Co., Ltd., Beijing, China] . Genotyping was conducted using the polymerase chain reaction-ligase detection reaction (PCR-LDR) method using an ABI 9600 system (Applied Biosystems, Foster City, CA, USA) (Wang et al., 2014) . Cycling parameters were as follows: 94°C for 2 min; 35 cycles of 94°C for 15 s; 60°C for 15 s; and 72°C for 30 s; and a final extension step at 72°C for 5 min. Two specific probes to discriminate the specific bases and one common probe were synthesized (Ramaniuk et al., 2014) . The common probe was labeled at the 3' end with 6-carboxy-fluorescein and phosphorylated at the 5' end. The reaction conditions for the LDR were: 94°C for 2 min, 20 cycles of 94°C for 30 s, and 60°C for 3 min. After the reaction was completed, an aliquot (1 mL) LDR products were mixed with 1 mL ROX passive reference dye and 1 mL loading buffer, denatured at 95°C for 3 min, and chilled rapidly in ice water. The fluorescent products of LDR were differentiated using an ABI sequencer 377 (Applied Biosystems).
Abi 377 sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 377 sequencer is a DNA sequencing instrument manufactured by Thermo Fisher Scientific. It utilizes the Sanger sequencing method to determine the nucleotide sequence of DNA samples. The ABI 377 sequencer is capable of analyzing multiple samples simultaneously and producing DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Abi 9600 system, by Thermo Fisher Scientific (2 mentions)
Tianamp blood dna kit, by Tiangen Biotech (1 mentions)
Abi prism bigdye terminator, by Thermo Fisher Scientific (1 mentions)
Nitrocefin, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Megalign pro, by DNASTAR (1 mentions)
Staphtype, by Ridom (1 mentions)
Gentra puregene extraction kit, by Qiagen (1 mentions)
Accu prep pcr dna purification kit, by Bioneer (1 mentions)
Taq dna ligase, by New England Biolabs (1 mentions)
10 protocols using abi 377 sequencer
1
Genotyping of Blood Samples
2
Genomic DNA Extraction and Genotyping
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Blood samples (1 mL) were collected, and genomic DNA was extracted from white blood cells using the TIANamp Blood DNA Kit (TianGen Biotech (Beijing) Co., Ltd., China). Genotyping was conducted by the polymerase chain reaction-ligase detection reactions (PCR-LDR) method using the ABI 9600 system (Applied Biosystems, Foster City, CA, USA) (Hu et al., 2014; Wang et al., 2014c) . Cycling parameters were as follows: 94°C for 2 min; 35 cycles of 94°C for 20 s; 56°C for 20 s; 72°C for 40 s; and a final extension step at 72°C for 3 min. Two probes to discriminate the specific bases and one common probe were synthesized. The common probe was labeled at the 3'-end with 6-carboxy-fluorescein, and phosphorylated at the 5'-end. The reaction conditions for LDR were: 94°C for 2 min; 30 cycles of 94°C for 30 s; and 56°C for 3 min. After the reaction, 1 mL LDR products was mixed with 1 mL carboxy-Xrhodamine (ROX) passive reference and 1 mL loading buffer, denatured at 95°C for 3 min, and chilled rapidly in ice water. The fluorescent products of LDR were differentiated using ABI sequencer 377 (Applied Biosystems).
3
Identifying Mutations in fliF Gene
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It has been determined that SJW3060 carried mutation only in fliF gene (Yamaguchi, unpublished data). The original mutation of SJW3060 was determined by DNA sequencing analysis. The DNA fragment containing fliF was amplified by polymerase chain reaction (PCR) using two primers surrounding the fliF gene and the genome DNA prepared from SJW3060 as a template. DNA sequencing samples were prepared using the purified PCR product as a template, the appropriate sequence primers, and ABIPRISM big dye terminator (Applied Biosystems).
The second mutation sites of the revertants were determined by exhaustive survey of the DNA sequence of the regions indicated by the mapping results. The entire length of Region I, II, or III was amplified by PCR using HindIII site-conjugated primers and the genome DNA prepared from each revertant, and then the PCR products were cloned into pBR322. The inserted DNAs were digested by combinations of appropriate restriction enzymes, and the resultant fragments were cloned into pHSG395. DNA sequencing samples were prepared using the plasmids as a template, M13 and the appropriate sequence primers, and ABIPRISM big dye terminator. DNA sequencing was performed using an ABI377 sequencer (Applied Biosystems).
The second mutation sites of the revertants were determined by exhaustive survey of the DNA sequence of the regions indicated by the mapping results. The entire length of Region I, II, or III was amplified by PCR using HindIII site-conjugated primers and the genome DNA prepared from each revertant, and then the PCR products were cloned into pBR322. The inserted DNAs were digested by combinations of appropriate restriction enzymes, and the resultant fragments were cloned into pHSG395. DNA sequencing samples were prepared using the plasmids as a template, M13 and the appropriate sequence primers, and ABIPRISM big dye terminator. DNA sequencing was performed using an ABI377 sequencer (Applied Biosystems).
4
Characterizing bla NDM-like Isolates
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The β-lactamase content of all blaNDM-like-positive isolates was determined by isoelectric focusing (IEF). Bacterial extracts were obtained by sonication of bacterial cells suspended in 1% glycine buffer and clarified by centrifugation. Sonicated cell extracts were analyzed by IEF in polyacrylamide gels containing ampholytes (pH 3.5–9.5; AP Biotech, Piscataway, NJ). The separated β-lactamases were visualized by covering the gel with the chromogenic cephalosporin nitrocefin (0.2 mg/ml; Oxoid Ltd., Basingstoke, United Kingdom; Papagiannitsis et al., 2015 (link)).
On the basis of the IEF data, PCR detection of various bla genes was performed by the use of primers specific for blaTEM−1, blaOXA−1, blaSHV, blaCTX−M, and blaCMY, as reported previously (Pałucha et al., 1999 (link); Pérez-Pérez and Hanson, 2002 (link); Woodford et al., 2006 (link); Coque et al., 2008 (link)). Both strands of the PCR products were sequenced using an ABI 377 sequencer (Applied Biosystems, Foster City, CA).
On the basis of the IEF data, PCR detection of various bla genes was performed by the use of primers specific for blaTEM−1, blaOXA−1, blaSHV, blaCTX−M, and blaCMY, as reported previously (Pałucha et al., 1999 (link); Pérez-Pérez and Hanson, 2002 (link); Woodford et al., 2006 (link); Coque et al., 2008 (link)). Both strands of the PCR products were sequenced using an ABI 377 sequencer (Applied Biosystems, Foster City, CA).
5
PCR Amplification and Sequence Analysis
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The PCR products were purified using a commercial gel purification kit (QIAquick PCR Purification Kit, Qiagen, Venlo, The Netherlands). The sequencing process was accomplished by applying the dye terminal method in an ABI 377 sequencer (Applied Biosystems, Waltham, MA, USA). Sequencing results were interpreted through a query to the Gen Bank local alignment search tool (BLAST) (blast.ncbi.nlm.nih.gov/Blast.cgi?).
6
Molecular Typing of Staphylococcus aureus
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Spa genotyping PCR for amplification of the S. aureus protein A (spa) repeat region was performed according to a published protocol [16 (link)], for genetic lineage identification of the bacterial samples. The X region of the spa gene was amplified by PCR with primers 1095F (5′-AGACGATCCTTCGGTGAGC-3′) and 1517R (5′-GCTTTTGCAATGTCATTTACTG-3′). DNA was obtained from isolated S. aureus colonies according to the extraction protocol previously described. PCR products were submitted to gel electrophoresis to evaluate purity and estimate the amplicon size. DNA sequences were obtained with an ABI 377 sequencer (Applied Biosystems, Foster City, Calif.). The forward and reverse sequence chromatograms were analyzed and compared with previous sequences from the Ridom StaphType™ (Ridom GmbH, Muenster, Germany). The identified and reliable sequences were assigned a numerical code by the specific spa type repeats found. All the FASTA sequences obtained after sequencing were aligned by MegAlign Pro with Omega Clustal (DNA Star, Inc.). The dendrogram construction and cluster analysis were obtained using neighbor joining BIONJ method. One clone of MRSA strain was included as a control.
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Spa genotyping PCR for amplification of the S. aureus protein A (spa) repeat region was performed according to a published protocol [16 (link)], for genetic lineage identification of the bacterial samples. The X region of the spa gene was amplified by PCR with primers 1095F (5′-AGACGATCCTTCGGTGAGC-3′) and 1517R (5′-GCTTTTGCAATGTCATTTACTG-3′). DNA was obtained from isolated S. aureus colonies according to the extraction protocol previously described. PCR products were submitted to gel electrophoresis to evaluate purity and estimate the amplicon size. DNA sequences were obtained with an ABI 377 sequencer (Applied Biosystems, Foster City, Calif.). The forward and reverse sequence chromatograms were analyzed and compared with previous sequences from the Ridom StaphType™ (Ridom GmbH, Muenster, Germany). The identified and reliable sequences were assigned a numerical code by the specific spa type repeats found. All the FASTA sequences obtained after sequencing were aligned by MegAlign Pro with Omega Clustal (DNA Star, Inc.). The dendrogram construction and cluster analysis were obtained using neighbor joining BIONJ method. One clone of MRSA strain was included as a control.
7
Microsatellite Analysis of Plant Genetic Diversity
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In total, samples were collected from the root tissue of 250 adult individuals through active search (Table 1 ), in order to cover as much of each area as possible, avoiding the sampling of individuals geographically close to each other, aiming to sample the genetic pool of each collection area. Posteriorly, the DNA was obtained according to Doyle and Doyle (1987 ), and the genetic material was amplified with a total of 14 nuclear microsatellite fluorescent primer pairs (EE2/EE5/EE23/EE32/EE9/EE25/EE43/EE45/EE47/EE48/EE52/EE54/EE59/EE63) developed by Gaiotto, Brondani, and Grattapaglia (2001 ).
The amplification reactions were performed by PCR in a final volume of 13 µl containing 7.5 ng of genomic DNA, 1× buffer (10 mmol/L Tris‐HCl, 50 mmol/L KCl and 21.5 mmol/L MgCl, pH 8.3), 0.27 µmol/L of each primer, 2.0 mmol/L MgCl2, 0.25 mg/ml BSA, 0.25 mmol/L dNTP, and 1U of Taq DNA polymerase in ultrapure sterile water. The PCRs were performed under the following conditions: 96°C for 2 min; 34 cycles of 94°C for 1 min, primer‐specific annealing as per Gaiotto et al. (2001 , T°C) for 1 min, 72°C for 1 min; with a final elongation performed at 72°C for 7 min. The analysis of amplicons in a denaturing gel (7 mol/L urea) with 4% polyacrylamide (Long Ranger 50%—Cambrex) was performed in a multiplex system, in a semiautomatic ABI 377 sequencer (Applied Biosystems) by using virtual filter D.
The amplification reactions were performed by PCR in a final volume of 13 µl containing 7.5 ng of genomic DNA, 1× buffer (10 mmol/L Tris‐HCl, 50 mmol/L KCl and 21.5 mmol/L MgCl, pH 8.3), 0.27 µmol/L of each primer, 2.0 mmol/L MgCl2, 0.25 mg/ml BSA, 0.25 mmol/L dNTP, and 1U of Taq DNA polymerase in ultrapure sterile water. The PCRs were performed under the following conditions: 96°C for 2 min; 34 cycles of 94°C for 1 min, primer‐specific annealing as per Gaiotto et al. (
8
Loss of Heterozygosity Analysis of BAP1 in Tumor Samples
9
Genotypic Characterization of HCMV UL97 Gene
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The PCR amplifi ed products were purifi ed using the Accu-Prep PCR DNA Puri fi cation Kit (Bioneer, South Korea) according to the protocol recommended by manufacturer. Automated DNA sequencing was carried out using an ABI 377 sequencer (Applied Biosystems, Foster City, CA). DNA sequences were analyzed using BioEdit software (version 7.0.5.3) and compared with wild-type GCV-sensitive AD169 strain (accession number BK000394.5) as a reference. The UL97 sequences of the 49 clinical strains have been assigned GenBank accession numbers. MG978138 to MG978177 and MH185085 to MH185092.
10
Multiplex Genotyping by Ligase Detection
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All genotyping experiments were carried out by the Shanghai Biowing Applied Biotechnology Company (Shanghai, China) using the ligase detection reaction (LDR) method. Firstly, target DNA sequences were amplified using multiplex polymerase chain reaction (PCR). Then, samples were incubated with 1 µL proteinase K (20 mg/mL) at 70°C for 10 min before the reaction was stopped by heating to 94°C for 15 min. The ligation reaction for each sample was carried out in a final volume of 20 µL, containing 20 mM Tris-HCl, pH 7.6, 25 mM potassium acetate, 10 mM magnesium acetate, 10 mM dithiothreitol, 1 mM nicotinamide adenine dinucleotide, 0.1% Triton X-100, 10 µL multiplex PCR product, 1 pmol each discriminating probe, 1 pmol each common probe, and 0.5 µL Taq DNA ligase (40 U/µL; New England Biolabs, Ipswich, MA, USA). LDRs were performed using 40 cycles of 94°C for 30 s and 63°C for 4 min. The fluorescent products were detected using an ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA).
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