A PCR fragment spanning the ompA promoter was amplified using the 6-carboxyfluorescein (FAM)- and 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX)-labeled primers [FAM]ompAFL and [HEX]ompR. NceR protein binding to the labeled DNA probe and DNAse I digestion reactions were performed as described previously (44 (link)). Detection of the DNAse I digestion peaks was carried out in a 3730 capillary sequencer (Applied Biosystems, Waltham, MA, USA) by Azenta Life Sciences (Chelmsford, MA, USA), and the alignment of the corresponding electropherograms was generated using GeneMapper Software Version 4.0 (Applied Biosystems). Negative control reactions were done using bovine serum albumin (BSA) at the same mass concentration used for NceR. Phosphorylated MisR protein was prepared as previously described as a positive control (45 (link)). A DNA template was amplified with primers ompAFL and ompR to generate a sequence ladder for each strand as previously described (46 (link)). The final electropherograms of the sequencing reactions were horizontally aligned with those generated in the footprint using GeneMapper Software Version 4.0 (Applied Biosystems). As confirmation, the HEX/FAM-labeled DNA probe was subjected to Sanger sequencing using primer pompR.
Genemapper software version 4
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Netherlands
GeneMapper software version 4.0 is a DNA fragment analysis software developed by Thermo Fisher Scientific. It is designed to analyze and interpret DNA fragment data generated by capillary electrophoresis instruments.
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Lab products found in correlation
Abi prism 3130, by Thermo Fisher Scientific (6 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Hi di formamide, by Thermo Fisher Scientific (4 mentions)
Abi prism 3500 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Abi prism 3130 genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (3 mentions)
Genescan 500 liz size standard, by Thermo Fisher Scientific (3 mentions)
Abi prism 3730 genetic analyser, by Thermo Fisher Scientific (3 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Abi 3500 dna genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Pop 4 polymer, by Thermo Fisher Scientific (3 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Snapshot multiplex kit, by Thermo Fisher Scientific (2 mentions)
Abi 3730 sequencer, by Thermo Fisher Scientific (2 mentions)
Abi prism 3130xl, by Thermo Fisher Scientific (2 mentions)
Automated 3730 dna analyzer, by Thermo Fisher Scientific (2 mentions)
3730 capillary sequencer, by Thermo Fisher Scientific (2 mentions)
91 protocols using genemapper software version 4
1
NceR Protein Binding Assay using FAM/HEX-labeled ompA Probe
2
PCR Primer Analysis and Electrophoresis
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Analysis of published polymerase chain reaction (PCR) primers (Supplementary Table 1 ), identified PCR products that were used for electrophoresis with the default program on the automated GeneScan ABI PRISM™ 3730xl DNA Analyzer (ThermoFisher Scientific, Waltham, MA, USA). The sizes of the detected fragments were analyzed using GeneMapper software version 4.0 (ThermoFisher Scientific, Waltham, MA, USA).
3
STR Profiling of Cell Lines
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The GenePrint® 10 system (Promega Corporation, Fitch-burg, WI, USA) was used to amplify human genomic DNA extracted from the GC-030-35 cell lines. The amplification products were assessed using the ABI 3730xl automated DNA analyzer (Thermo Fisher Scientific). GeneMapper software version 4.0 (Thermo Fisher Scientific) was used to analyze the data, which were compared with the American Type Culture Collection (ATCC, Manassas, VA, USA), DSMZ cell culture collection, and the JCRB Cell Bank database for reference matching. STR testing was performed at GENEWIZ (South Plainfield, NJ, USA).
4
Genotyping Protocol for FTO rs9939609 Variant
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Whole genomic DNA was isolated from peripheral blood leukocytes according to a protocol from commercial DNA isolation kit (Macherey-Nagel, Germany). Identification of rs9939609 of FTO gene was performed by means of two methods: polymerase chain reactions, PCR (using Takara Amplification Kit, Japan), and minisequencing (using SnaPshot Multiplex Kit, Thermo Fisher Scientific, USA). The reactions were carried out in the presence of specifically designed pair of primers:
5'-CACTAACATCAGTTATGCAT-3' – forward primer
5'-CCATTTCTGACTGTTACCTA-3' – reverse primer
5
Microsatellite Instability Analysis Protocol
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MSI testing was performed only in cases of abnormal or dubious MMR IHC result. Genomic DNA was extracted from fixed, paraffin-embedded (FFPE) tumor tissue (tumor-rich areas > 50%) and normal tissue using the QIAamp DNA Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Concentration of the extracted DNA was assessed by real time polymerase chain reaction (PCR) using the Quantifiler® Human DNA Quantification kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Microsatellite instability (MSI) analysis was performed through PCR reaction, using the CC-MSI kit (AB Analitica, Padova, Italy) according to the manufacturer’s protocol. This kit is designed to co-amplify 10 markers (BAT25, BAT26, D2S123, D5S346, D17S250, NR21, NR24, BAT40, TGFbRII and D18S58) in two separate reactions. The fluorescent amplified PCR products (from normal and tumor tissue) were analyzed by capillary gel electrophoresis on an ABI 3730XL DNA Analyzer (Thermo Fisher Scientific), using the GeneMapper software, version 4.0 (Thermo Fisher Scientific). Tumors were classified as MSI-high (MSI-H; 4/10 markers showing MSI), MSI-low (MSI-L; 1–3/10 markers showing MSI) or MS stable (MSS; no markers showing instability).
6
Establishing Drug-Resistant Cell Lines
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PLX4032-resistant (PLX-R) and dimethyl sulfoxide-resistant (DMSO-R) MeOV and MeTA cells were selected by treating the whole population of parental cells with increasing concentrations of PLX4032 or DMSO. DMSO-R cells were sensitive to PLX4032 and were used as a comparative model to exclude a toxic effect of DMSO chronic treatment or its potential interference with the effects exerted by PLX4032. Briefly, to obtain drug-resistant cell populations, the cells were seeded twice a week at a density of 1.5 x 106 and treated over six months with PLX4032 (250 nM- 1.5 µM) or with the corresponding volume of DMSO. The authenticity of the selected cells was checked by Short Tandem Repeat (STR) profile analysis performed by the Immunohematology and Transfusion operative unit, IRCCS Ospedale Policlinico San Martino, Genoa. Fifteen highly polymorphic STR loci plus amelogenin (Cell IDTM System, Promega, Milan), were used. Detection of amplified fragments was obtained by ABI PRISM 3100 Genetic Analyzer. Data analysis was performed by GeneMapper® Software version 4.0 (Thermo Fisher Scientific, Waltham, MA, USA).
7
Genetic Polymorphism Analysis of Stress-Related Genes
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Genetic polymorphism of HTR1A, BDNF, and glucocorticoid receptor (FKBP5) were assessed. For genotyping, genomic DNA was isolated from EDTA-pretreated venous blood samples. The DNA fragments of interest were amplified by using a polymerase chain reaction (PCR). Three SNPs, rs6295 in HTR1A,7 (link) rs6265 in BDNF,22 (link) rs9296158 in FKBP5,11 (link) were selected from among HTR1A, BDNF, FKBP5 variants identified in dbSNP (http://www.ncbi.nlm.nih.gov/SNP ) as having functional significance. These three SNPs were previously investigated with respect to vulnerability to emotional distress and reduction of QoL level. The genotyping was screened with a single-base primer extension assay using an ABI PRISM® SNaPshot™ Multiplex Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Analyses were performed using Genemapper Software version 4.0 (Thermo Fisher Scientific).
8
Amplification and Analysis of cMS Loci
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In order to amplify the cMS loci, primers were either obtained from the SelTarbase (Version 201307, www.seltarbase.org )27 (link) or designed using the primer3 software (Primer3web version 4.0.0, http://primer3.ut.ee/ ), with one primer of the primer set carrying a 5′ fluorescent (fluorescein isothiocyanate) label. Primers were designed to generate amplicons in range between 100 and 150 nucleotides for robust PCR amplification (Supplementary Table 6 ). PCR was performed in a total volume of 5 µl containing 0.5 µl 10× reaction buffer (Invitrogen, Karlsruhe, Germany), 1.5 mM MgCl2, 200 mM dNTP mix, 0.3 mM of each primer, 0.1 U Taq DNA polymerase (Invitrogen), and 10 ng of genomic DNA, using the following protocol: initial denaturation at 94 °C for 5 min; 36 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 45 s, and primer extension at 72 °C for 1 min; final extension step at 72 °C for 7 min. PCR fragments were separated on an ABI3130xl genetic analyzer (Applied Biosystems, Darmstadt, Germany). Generated raw data were analyzed using the GeneMapper™ Software version 4.0 (ThermoFisher, Waltham, USA). Peak height profiles were extracted and processed using ReFrame based on R version 3.4.3.
9
Genotyping of Botrytis cinerea strains from agricultural and non-agricultural habitats
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A subset of 109 strains collected from non-agricultural habitats were genotyped (Table 1 ). They were compared to 327 agricultural strains sampled from lettuce and tomato plants grown in several greenhouses in the South of France (Leyronas et al., 2015a ,b (link),c (link)).
Genomic DNA was extracted from aliquots of 15 mg lyophilized fungal material (harvested from two-week old cultures on Potato Dextrose Agar), following the DNeasy Plant extraction Kit protocole (Qiagen). The nine microsatellite markers designed for B. cinerea by Fournier et al. (2002) (link) were amplified following the protocol described by Leyronas et al. (2015b) (link). To determine the size of the microsatellites, the PCR products were scanned with the help of an ABI 3730 sequencer (Applied Biosystems). GeneMapper software version 4.1 (Applied Biosystems) was then used for the microsatellite size analysis. Complete microsatellite size profiles (referred to as “haplotypes” hereafter) were obtained for 109 environmental strains and 327 agricultural strains.
Genomic DNA was extracted from aliquots of 15 mg lyophilized fungal material (harvested from two-week old cultures on Potato Dextrose Agar), following the DNeasy Plant extraction Kit protocole (Qiagen). The nine microsatellite markers designed for B. cinerea by Fournier et al. (2002) (link) were amplified following the protocol described by Leyronas et al. (2015b) (link). To determine the size of the microsatellites, the PCR products were scanned with the help of an ABI 3730 sequencer (Applied Biosystems). GeneMapper software version 4.1 (Applied Biosystems) was then used for the microsatellite size analysis. Complete microsatellite size profiles (referred to as “haplotypes” hereafter) were obtained for 109 environmental strains and 327 agricultural strains.
10
Molecular Typing of Mycobacterium tuberculosis
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The isolates were cultured on Löwenstein Jensen medium and spoligotyping was used to characterize the polymorphic direct repeat region of the Mtb chromosome [5] (link). RFLP typing was performed using the insertion sequence IS6110 as a probe and PvuII as the restriction enzyme [6] (link), [7] (link). Finally, standardized 24-loci MIRU-VNTR [1] (link) was performed using the MIRU-VNTR typing kit (Genoscreen, Lille, France). The PCR products were run with 1200 LIZ size standard on ABI3131×l sequencers with 16 capillaries. Sizing of the PCR-fragments and assignments of MIRU-VNTR alleles were done with the GeneMapper software version 4.1 (Applied Biosystems) according to the instructions of the manufacturer. External quality assurance for MIRU-VNTR was provided by the European Centre for Disease Prevention and Control (ECDC) together with the Dutch National Institute for Public Health and the Environment (RIVM) as well as by the TB PAN-NET project.
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