Following treatment, DNA was extracted from cells using the DNeasy Blood and Tissue Kit (69 504, Qiagen). A 5ng aliquot of cellular DNA was used for each real-time quantitative PCR (qPCR). The abundance of mtDNA and nDNA was determined using a CFX96 real-time PCR detection system (Bio-Rad). Measurements were performed in duplicate with 100 n m of both sense and antisense primers in a final volume of 20 µL, using iQTM SYBR Green Supermix (1 708 880, Bio-Rad). Primers and PCR conditions are listed in Table S2A. PCR products were sequenced with the BigDye terminator kit (PerkinElmer Applied Biosystems), using an ABI3100 capillary sequencer (PerkinElmer Applied Biosystems). Relative abundance of mitochondrial genes over ACTB was calculated using the 2−ΔΔCT formula.
Abi 3100 capillary sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 3100 capillary sequencer is a DNA sequencing instrument that utilizes capillary electrophoresis technology to analyze genetic samples. It is designed to perform high-throughput, automated DNA sequencing with high accuracy and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator kit, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Iqtm sybr green supermix, by Bio-Rad (1 mentions)
Qiamp dna blood mini kit, by Qiagen (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Seqman tool, by DNASTAR (1 mentions)
Abi prism 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Coffalyser software, by MRC-Holland (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Masterpure complete dna purification kit, by Illumina (1 mentions)
Seqman software, by DNASTAR (1 mentions)
Sephadex g 50 dna grade fine, by Cytiva (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
10 protocols using abi 3100 capillary sequencer
1
Quantifying Mitochondrial DNA Abundance
2
Quantifying CEL VNTR Repeats
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Genomic DNA was extracted from peripheral blood leukocytes according to a standard protocol (QIAmp DNA Blood Mini kit (Qiagen Cat No./ID 51106) or MagNA Pure isolation kit (Roche Cat# 03730964001)). For analysis of CEL VNTR lengths, polymerase chain reaction (PCR) followed by DNA fragment analysis was performed as described in [25 (link)]. Briefly, PCR was carried out using a forward primer binding upstream and partly into the first repeat of the VNTR region, and a NED-labelled reverse primer binding downstream of the VNTR, resulting in an amplified product covering the complete VNTR. The PCR products were diluted and fragment analysis performed on an ABI 3100 capillary sequencer (Applied Biosystems). In addition to a DNA size standard, a set of predetermined samples was used for calibration of VNTR lengths. In the resulting spectra, the size (base pairs) of the NED-labelled DNA fragments determined the total number of the CEL VNTR repeats.
3
Detecting p53 Mutations in Breast Cancer
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To consider breast cancer subtype, we utilized data on p53 mutation status of the tumor. Archived paraffin-embedded tumor tissue was obtained and DNA was extracted (n = 859). Mutations were detected in exons 5-8 of p53, described in detail in Rossner et al. [23 (link)]. Samples were amplified using polymerase chain reaction (PCR) and the Surveyor Mutation Detection Kit (Transgenomic, Omaha, NE, USA) was used to screen for p53 mutations. Positively identified samples were confirmed and identified using PCR amplification and sequencing using an ABI 3100 capillary sequencer (Applied Biosystems Inc., Foster City, CA, USA).
4
Malaria Detection and Identification Protocol
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DNA was extracted from 200 μl of thawed EDTA-blood or four 3 mm DBSs with the TIANamp Blood or Blood Spots DNA Kits (TIANGEN, Beijing, CHINA) according to the manufacturer’s instructions. The extracted DNA was dissolved in 35 μl ddH2O and quantified with NanoDrop (Thermo Fisher, Waltham, USA) and stored at – 20 °C. The standard quantitative TaqMan PCR (qPCR) was performed on 40 ng DNA with a LightCycler® 96 (Roche), the genus qPCR primers were derived from the reference [31 (link)] and included a forward primer (5′-GTTAAGGGAGTG AAGACGA TCAGA-3′), a reverse primer (5′-AACCCAAAGACTTTGATTTC TCATAA-3′) and probe (5′-FAM-ACCGTCGTAA TCTTAACCAT AAACTATGCC GAC TAG-TAMRA-3′). P. falciparum and P. vivax assays were performed in two separate reactions as reported [32 (link)]. At least three positive and negative controls were included to each experiment. Samples with a cycle threshold (CT) ≤ 40 were considered positive.
For species identification by sequencing, DNA was amplified with the genus primers [31 (link)], and subjected to Sanger sequencing on an ABI3100 capillary sequencer (Applied Biosystems, Waltham, USA).
For species identification by sequencing, DNA was amplified with the genus primers [31 (link)], and subjected to Sanger sequencing on an ABI3100 capillary sequencer (Applied Biosystems, Waltham, USA).
5
Variant Verification in PALB2 and CHEK2 Genes
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DNA variants, including single-nucleotide variants (SNPs), small ins/del, and CNVs, identified through the method tested in the current study in the PALB2 and CHEK2 genes, were further verified using a second independent molecular technique. Specifically, PCR-specific amplification followed by Sanger sequencing was performed for point variants and/or small ins/del. After amplicon verification on a 2% agarose gel, sequencing was carried out using an ABI 3100 capillary sequencer (Applied Biosystems Inc., Foster City, CA, USA). The obtained electropherograms were analyzed using the SeqMan tool (DNASTAR, Inc., Madison, WI, USA). For CNV validation, multiplex ligation probe amplification (MLPA) was carried out using gene-specific SALSA MLPA probe sets (MRC-Holland, Amsterdam, the Netherlands) and an ABI PRISM 3130 XL genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA) for fragment separation. Finally, the Coffalyser software (MRC-Holland, Amsterdam, the Netherlands) was used for MLPA results analysis, according to the manufacturer’s instructions.
6
PCR Amplification and Sequencing of Tumor DNA
7
Multiplex PCR for DNA Fingerprinting
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For DNA fingerprinting analysis, multiplex PCR reactions were performed by amplifying 1 ng of genomic DNA using the genRESH MPX-2 and genRESH MPX-3 kits (Serac GmbH, Bad Homburg, Germany). Amplified products were analyzed on an ABI 3100 capillary sequencer and profiled by the genotyper V3.10 software (Applied Biosystems, Carlsbad, CA, USA).
8
Sequencing Protocols for Alzheimer's Genes
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The coding regions and the boundary intronic regions of APP, PSEN1 and PSEN2 genes we sequenced by direct Sanger sequencing, and 17, 11, and 10 primer pairs were designed to amplify the above-mentioned regions of APP, PSEN1, and PSEN2, respectively (Table S1 ). Genomic DNA was obtained by a pellet of at least 5 × 105 cells using the MasterPure Complete DNA purification kit (Epicentre Biotechnologies, Madison, WI, USA) following the manufacturer’s instructions.
Each amplicon was individually amplified, assessed for quality on 2% agarose gel, and purified before sequencing reactions. Direct sequencing was performed with the ABI 3100 capillary sequencer (Applied Biosystems Inc., Foster City, CA, USA), and sequence data analysis was carried out using the SeqMan software (DNASTAR, Inc., Madison, WI, USA). Next, DNA variants were categorized accordingly to Ensembl (https://www.ensembl.org/index.html ), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ ) and dbSNP (https://www.ncbi.nlm.nih.gov/SNP/ ) databases and the possible impact of specific variations at the protein level was predicted using VarSome (https://varsome.com ) and or Human Splicing Finder (http://www.umd.be/HSF/HSF.shtml ) tools.
A full list of the primer pairs used to amplify APP, PSEN1 and PSEN2 genes are illustrated inTable S1 .
Each amplicon was individually amplified, assessed for quality on 2% agarose gel, and purified before sequencing reactions. Direct sequencing was performed with the ABI 3100 capillary sequencer (Applied Biosystems Inc., Foster City, CA, USA), and sequence data analysis was carried out using the SeqMan software (DNASTAR, Inc., Madison, WI, USA). Next, DNA variants were categorized accordingly to Ensembl (
A full list of the primer pairs used to amplify APP, PSEN1 and PSEN2 genes are illustrated in
9
Quantitative RT-PCR for Macrophage Gene Analysis
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Total RNA from macrophages was extracted with RNAqueousR-Micro kit (Ambion) following the manufacturer’s protocol and reverse-transcribed into cDNA with iScript cDNA Synthesis Kit (Bio-Rad). Changes in target genes mRNA levels were determined by relative RT-qPCR with a CFX96 Real-Time PCR Detection System (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad) detection of single PCR product accumulation. RT-qPCR analyses were performed in duplicate with 100nM of both sense and anti-sense primers in a final volume of 20μl using iQ SYBR Green Supermix (Bio-Rad). Specific primers were designed using Primer3 (S1 Table ). PCR conditions were 95°C for 3min followed by 40 cycles of 15sec at 95°C, 30sec at 60°C. The PCR products were sequenced with the BigDye terminator kit (Perkin Elmer Applied Biosystems). The multiScreen SEQ384 Filter Plate (Millipore) and Sephadex G-50 DNA Grade Fine (Amersham Biosciences) dye terminator removal were used to purify sequence reactions before analysis on an ABI3100 capillary sequencer (Perkin Elmer Applied Biosystems). The efficiency of each set of primers was determined by dilution curves (S1 Table ). The relative changes in target gene/GAPDH mRNA ratio were determined by the formula: 2 ∆∆ct [31 (link)] (The MIQUE Guidelines, 2009).
10
Dipterocarp Molecular Identification Protocol
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PCR was performed in a 40-µL reaction volume containing 4 µL PCR 10X buffer, 1 µL 25 mM dNTP, 1 µL 20 µM of each primer, 1 µL 25 mM MgCl 2 , 1 µL BSA, 1 µL 2.5 U Taq DNA polymerase and approximately 50 ng genomic DNA. The rbcL, matK, and trnH-psbA genes from Dipterocarp samples were amplified using a standard protocol (Table 3). The amplification conditions for the two rbcL fragments (rbcLa and rbcLc), each approximately 700 bp in length, and for the 300-bp fragment of trnH-psbA, were 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 48°C for 30 s, and 72°C for 1 min. The reaction was completed by a 10-min extension and hold at 4°C. The amplification conditions for the 900-bp matK fragment were: denaturation at 95°C for 5 min; 30 cycles of 40 s at 95°C, 40 s at 52°C and 1 min at 72°C, followed by extension at 72°C for 10 min and hold at 4°C. The identities of the PCR products were verified by electrophoresis on 0.8% agarose gels. All PCR products were purified using a QIA quick PCR purification kit (Qiagen, Germany). The purified PCR products were sequenced in both directions with the same primers as for PCR using a Bigdye terminator v3.1 cycle sequencing kit (Applied Biosystems) on an ABI 3100 capillary sequencer following the manufacturer instructions.
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