Genomic DNA was extracted from peripheral blood by phenol–chloroform method. The genotype of each tag SNP for COPD patients and healthy smokers was screened by SNaPshot according to the manufacturer’s protocol (Thermo Fisher Scientific). In brief, multiplex PCR was performed by primers given in Supplementary Table 1 with FastStart Taq DNA polymerase (Roche, Basel, Switzerland). After alkaline phosphatase (Shrimp, Takara-Bio Inc., Kusatsu, Japan) and exonuclease I (Takara Bio Inc.) clean-up, single base extension was performed by SNaPshot Multiplex Ready Reaction Mix (Thermo Fisher Scientific) and the products were analyzed on ABI PRISM 3730 sequencer (Thermo Fisher Scientific). The genotypes of some random samples were confirmed by resequencing in 3730 sequencer.
Abi prism 3730 sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The ABI PRISM 3730 is a DNA sequencer developed by Thermo Fisher Scientific. It is designed to perform high-throughput DNA sequencing. The core function of the ABI PRISM 3730 is to analyze DNA samples and generate DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Genemapper v4, by Thermo Fisher Scientific (3 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (3 mentions)
Exonuclease 1, by Takara Bio (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Genescan 500 liz size standard, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Snapshot, by Thermo Fisher Scientific (1 mentions)
Faststart taq dna polymerase, by Roche (1 mentions)
Kapa2g fast pcr kit, by Roche (1 mentions)
Kod plus polymerase, by Toyobo (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Universal genomic dna extraction kit, by Takara Bio (1 mentions)
Agarose gel dna purification kit, by Takara Bio (1 mentions)
Ptc 100 thermocycler, by Bio-Rad (1 mentions)
Bigdye terminator 3, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Thermo Fisher Scientific (1 mentions)
Genemarker v2, by SoftGenetics (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Abi prism bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Geneamp xl pcr kit, by Thermo Fisher Scientific (1 mentions)
25 protocols using abi prism 3730 sequencer
1
Genotyping COPD SNPs in Smokers
2
Genetic Variant Identification in Familial Disorder
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Venous blood (3.5 ml) was collected form the proband and his parents. Genomic DNA was extracted using QIAamp DNA Mini kit (Qiagen GmbH) and used for Sanger sequencing. The data from high-throughput sequencing were used to design primers for suspected pathological variants using PRIMER3 (version 0.4.0, http://bioinfo.ut.ee/primer3-0.4.0/ ), (forward, 5′-CGTGATTGTAGCACTTGCCTG-3′ and reverse, 5′-GAATGTGGACCCTGTTGTGTG-3′). PCR was performed using KAPA 2G Fast PCR kit (Roche, cat. no. KK5009), as follows: Initial denaturation at 95°C for 5 min, followed by 32 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 45 sec and final extension at 72°C for 5 min. PCR products (length, 412 bp) were analyzed by gel electrophoresis and purified. Capillary electrophoretic sequencing was performed using an ABI PRISM 3730 sequencer (Thermo Fisher Scientific, Inc.) and variants were analyzed. The Sanger sequencing results were aligned with SALL4 reference sequence (NM_020436, http://www.ncbi.nlm.nih.gov/nuccore/NM_020436 ) using SeqMan 7.1 software (16 (link)).
3
Bovine Chemerin Gene Sequence Analysis
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According to the sequence of the bovine chemerin gene (GenBank accession no. NM_001046020), two pairs of primers were designed to amplify a coding region of the chemerin gene that included exons 2 to 4. The gene primer sequences were: E1-forward: 5′-GGCTGGGCTAAGGAACAGTG-3′, E1-reverse:5′-GGTCTCCAACCTCAGGCTTC-3′. E2-forward: 5′-CTGCAGGATAGTTCTGACTTTTG-3′, E2-reverse: 5′-GCTTTATTAGCTCAGGGGTCA-3′. Polymerase chain reaction (PCR) amplifications were performed in 30-μL reaction mixtures, each containing 10 to 30 ng DNA template, 5 μM of each primer, 3 μL 10×PCR Buffer for KOD-Plus-, 0.20 mM dNTPs, 1.67 mM MgSO4, and 0.5 U KOD-Plus- Polymerase (Toyobo, Osaka, Japan). The PCR protocol was 94°C for 9 min followed by 35 cycles of 30 s at 94°C, 30 s at 63°C or 60°C, and 1 min at 72°C and a final extension at 72°C for 5 min. Next, the PCR products were electrophoresed through 1.5% agarose gel and single band was confirmed. Then, the products were purified using Fast Gel/PCR Extraction Kit (Nippon Genetics Co, Ltd, Tokyo, Japan). Finally, the PCR products were sequenced to detect the presence of SNPs. Bi-directional DNA sequencing of PCR amplicons was carried out using an ABI PRISM 3730 sequencer (Applied Biosystems, Foster, CA, USA). Sequencing variants were detected by visual examination of the sequencing map followed by alignment using CLUSTAL (Higgins and Sharp, 1988 (link)).
4
Myxozoan SSU rRNA Gene Amplification and Sequencing
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Ethanol-preserved plasmodia were used for genomic DNA extraction according to the protocols recommended by the manufacturer of the TIANamp Genomic DNA Kit (Beijing Tiangen Biotech Co. Ltd., China). The SSU rRNA gene was amplified with universal eukaryotic primer pairs 18e [15 (link)] and 18r [16 (link)], for spores collected in 2015 (narrow type), or MyxospecF [17 (link)] and SphR [18 (link)], for spores collected in 2013 (wide type). The next step of PCR, purification, cloning and transformation were performed following the procedure of Zhao et al. [19 (link)]. DNA sequences were then defined bidirectionally by Sanger sequencing on an ABI PRISM® 3730 sequencer (Applied Biosystems Inc., Foster, USA), with the resulting contiguous sequences assembled by the Lasergene package v 5. 05 (DNASTAR, Madison, Wisconsin) and corrected with BioEdit 7.2.5 [20 ] based on the original sequence chromatograms. Contiguous sequences were then submitted to an NCBI BLASTn search for comparison with other myxozoan sequences.
5
Chloroplast DNA Extraction and Sequencing
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Total genomic DNA was isolated from silica gel–dried leaf material using a Universal Genomic DNA Extraction Kit (Takara, Dalian, China). Five chloroplasts (the rbcL gene; the rps16 intron; the trnT–F region; the atpB–rbcL and psbA–trnH intergenic spacers) were selected for phylogenetic inference. For trnT–F regions, primers A (or A1) and D, as well as c and f as in Taberlet et al.92 (link) and Bremer et al.93 (link) were used with the internal. For rbcL, primers Z1 and 3′995 (link) were used. The atpB-rbcL and psbA-trnH spacers were amplified and sequenced using the primers as described by Manen et al.96 (link) and Sang et al.97 (link), respectively. The rps16 intron was amplified and sequenced with primers F and 2 R98 (link),99 (link). All polymerase chain reactions (PCRs) were run in a PTC–100 thermocycler (MJ Research, Ramsey, MN, USA). PCR products were purified using an agarose gel DNA purification kit (Takara, Shiga, Japan), following the manufacturer’s instructions. Sequencing was performed with BigDye Terminator 3.1 (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 3730 Sequencer using the same primers as employed for the PCR amplifications. All sequences were analyzed and assembled with Sequencher ver.4.14 (Gene Code, Ann Arbor, MI, USA).
6
Genomic Analysis of Venetoclax Resistance
7
Isolation and Identification of Gut Bacteria from Drosophila Larvae
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Five D. melanogaster guts from 3rd instar larvae were homogenized in 200 μl of PBS, and spread onto MRS, LB, and 869 agar plates. 869 agar is a reduced nutrient medium comprised of 0.04 g CaCl2–2H2O, 0.1 g glucose, D+, 0.5 g NaCl, 1 g tryptone, 0.5 g yeast extract, and 15 g agar in 1000 ml of diluted water. All plates were incubated for 72 h at 30 °C. A. pomorum, L. fructivorans and the other bacteria were identified by sequencing. Briefly, DNA was isolated from colonies on MRS plates with typical characteristics of Acetobacter and Lactobacillus bacteria [43 ] and used as template to PCR amplify products generated using universal 16 S rRNA 27 F and 1522 R primers (Supplementary Table 2 ). The same DNA isolation and PCR procedures were used for randomly selected colonies on LB agar plates and 869 agar plates. Purified PCR products were Sanger sequenced using an ABI Prism 3730 sequencer (Applied Biosystems). Resulting sequences were then referenced to the GenBank database by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi ).
8
Multiplex PCR with SSR Markers
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Twenty-three simple sequence repeat (SSR) markers were screened from a list of 76 SSRs previously developed by Li et al.30 and analysed using three sets of multiplex PCR reactions in this study (Supplementary Table S6 ). Each multiplex set was carefully assembled based on the compatibility of SSRs during the PCR and on the molecular size of their amplicons. Forward primers of SSR markers were labelled with one of the following fluorescent dyes: carboxy fluorescein (FAM), carboxytetrame thylrhodamine (TAMRA) or hexachloro-6-carboxy fluorescein (HEX). The reaction mixtures (10 μL) contained 0.2 μL template DNA, 0.1 μL of each primer, and 5 μL Multiplex PCR Master Mix (QIAGEN Multiplex PCR Kit, Qiagen, Germany). The PCR reactions were performed in a Life Pro thermocycler with an initial denaturation step of 5 min at 95 °C, followed by 20 cycles of 95 °C for 30 s, 55–58 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for 10 min. For PCR fragment size determinations, 0.5 μL of an internal size standard (Liz-500, LIZ) was mixed with 0.3 μL of PCR product and 9.5 μL formamide. The mixture was heated to 95 °C for 5 min, then cooled down on ice, and finally subjected to fragment analysis on an ABI PRISM 3730 sequencer (Applied Biosystems, Unites States of America). The alleles were sized using genemarker v2.2.0 (SoftGenetics).
9
Precise Breakpoint Mapping via PCR
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Junction fragments across the t(3;4) breakpoint were amplified by long-range PCR using the GeneAmp® XL PCR Kit (Life Technologies). Products from these reactions were purified by incubation with 10 units of Exonuclease I and 0.5 unit of SAP at 37 C during 45 min followed by heat inactivation (80 C during 30 min). They were then sequenced using the ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3730 sequencer (Applied Biosystems, Foster City, CA, USA). Computational analysis of the resulting sequences allowed precise localization of the breakpoints on SSC3 and 4.
10
Felid DNA Extraction and Genotyping
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Procedures for DNA extraction and genotyping are outlined in detail in [11 (link)]. Briefly, after DNA extraction from tissue samples with the DNeasy 96 Blood and Tissue kit (Qiagen), samples were genotyped at 21 short-tandem repeat loci (STRs) [53 (link), 54 (link)] in seven multiplexes [11 (link)]. Compared to [11 (link)], the use of two STR markers (Fca078 and Fca001) was discontinued in the present study due to time-consuming allele scoring. Molecular sex determination was done by amplifying regions on the zinc finger region on the felid X- and Y-chromosome with primers developed by [55 (link)]. Amplification reactions contained 5.0 μl 2x Multiplex PCR MasterMix (Qiagen), 1.0 μl Primer mix (see Table 1 in [11 (link)] for concentrations of each primer set), 0.05 μl BSA (NEB), and 2.95 μl ddH2O in a 10 μl PCR reaction with 1μl template DNA. The PCR thermocycling protocol included the following steps: 10 min at 95°C followed by 29 cycles of 30 s at 94°C, 30 s at 56/57/58/59°C (annealing temperature differed among multiplex sets, see [11 (link)]), 1 min at 72°C, with a final elongation step of 45 min at 72°C. An ABI PRISM 3730 sequencer was used to analyse samples and the GeneMapper v4.1 (Applied Biosystems) software program was used for subsequent genotyping. For assessment of genotyping reliability, 10% of the samples were chosen randomly and analysed a second time.
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