The ribosome binding reaction mixtures were prepared on ice in a total volume of 6 μl containing 50% Nuclease-treated rabbit reticulocyte lysate (Promega), 20 pmol of primer (5′-6-FAM-AATTGTTCCAGGAACCAG-3′), 20 μM amino acid mixture minus methionine, 20 μM amino acid mixture minus leucine,0.4U/μL RNaseOUT (Invitrogen) and 50 mM Tris-HCl, pH 7.5. Reactions were added with or without 1 mg/mL CHX and incubated at 37°C for 5 min. Then the reaction mixtures were added with 0.5 μg of mRNA, followed by incubating at 30°C for 20min to allow the translation machinery to assemble. The reverse transcriptase reaction was performed in a total volume of 20 μl containing entire ribosome binding reaction, 1x Superscript III reverse transcriptase buffer, 5 mM DTT, 1 mg/ml cycloheximide, 500 μM of each of four dNTPs, 1.5U/μL RNaseOUT, 5U/μL Superscript III reverse transcriptase and incubated at 25°C for 10 min. Reactions were terminated by extracting with phenol:chloroform followed by ethanol precipitation. Primer extension products were resuspended in 10 μL of Hi-Di™ formamide. A 2 μL aliquot was run with 0.2 μL GeneScan™ 500 LIZ™ dye Size Standard (Fisher) on an ABI 3730xl instrument. Data were analyzed by GeneMapper software (Life Technologies).
Genescan 500 liz dye size standard
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneScan 500 LIZ dye Size Standard is a DNA size standard used for accurate sizing of PCR fragments and DNA sequencing samples. It contains a set of DNA fragments of known sizes labeled with the LIZ fluorescent dye, allowing for precise identification of fragment sizes when used in conjunction with capillary electrophoresis instrumentation.
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Lab products found in correlation
Hi di formamide, by Thermo Fisher Scientific (7 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (4 mentions)
Genemapper software, by Thermo Fisher Scientific (3 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
Nuclease treated rabbit reticulocyte lysate, by Promega (2 mentions)
Rnaseout, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Promega (2 mentions)
Abi 3130xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
A pcr clean up system, by Promega (1 mentions)
Wizard sv gel, by Promega (1 mentions)
Genemapper v5, by Thermo Fisher Scientific (1 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Abi prism 3730xl dna analyser, by Thermo Fisher Scientific (1 mentions)
Abi 3730 sequencer, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcriptase kit, by Promega (1 mentions)
Human xpressref universal total rna, by Qiagen (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Abi 3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
26 protocols using genescan 500 liz dye size standard
1
Ribosome Binding Reaction and RT-PCR Analysis
2
Ribosome Binding Reaction Profiling
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The ribosome binding reaction mixture was prepared on ice in a total volume of 10 μl containing 50% Nuclease-treated rabbit reticulocyte lysate (Promega), 20 pmol of primer (5ʹ- 6-FAM-AATTGTTCCAGGAACCAG- 3ʹ), 20 μM amino acid mixture, 0.4 U/μl RNaseOUT (Invitrogen) and 50 mM Tris-HCl pH 7.5. Reactions were treated with 0.5 mg/ml CHX followed by incubation at 37 °C for 5 min. After addition of 0.3 mg of reporter mRNAs, the reaction mixtures were incubated at 30 °C for 20 min to allow the translation machinery to assemble at the start codon. The reverse transcriptase reaction was conducted in a total volume of 20 μl containing the entire ribosome binding reaction, 1x Superscript III reverse transcriptase buffer, 5 mM DTT, 40 mM KCl, 3 mM MgCl2, 50 mM Tris-HCl pH7.5, 0.5 mg/ml cycloheximide, 0.8 mM of dNTP, 1.5 U/μl RNaseOUT, 5 U/μl Superscript III reverse transcriptase. After incubation at 25 °C for 10 min, the reaction was terminated by nucleic acids extraction by phenol:chloroform and ethanol precipitation. The primer extension products were resuspended in 10 μl of Hi-Di formamide. 2 μl aliquot was run with 0.2 μl GeneScan 500 LIZ dye Size Standard (Fisher) on an ABI 3730xl instrument. Data is analyzed by Peak Scanner 2 software.
3
DNase I Footprinting of Bacterial Genes
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The DNase I footprinting assay was performed by following a protocol developed by Wang et al. (2012 (link)). Briefly, the promoters of the arlR, ica, and pitR genes were cloned into a pUC18B-T vector (Shanghai Biotechnology Corporation, China), and the plasmids were used as the template for preparation of fluorescein amidite (FAM)-labeled probes with the primers M13F and M13R (both FAM-labeled). The FAM-labeled probes were purified using Wizard SV Gel and a PCR Clean-Up System (Promega, Southampton, UK), and quantified using NanoDrop 2000C (Thermo Scientific). For the DNase I footprinting assay, 200 ng probes were incubated with different amounts of r-YycF in 40 μl of binding buffer at 30°C for 30 min. Subsequently, 10 μl DNase I (0.01 unit) (Promega, UK) and 100 nmol CaCl2 were added, incubated for 1 min at 25°C, and stopped using 140 μl DNase I stopping solution (200 mM unbuffered sodium acetate, 30 mM EDTA, and 0.15% SDS). The DNA samples extracted with phenol/chloroform and precipitated with ethanol, and the pellets were dissolved in 35 μl MilliQ water. The samples were loaded onto a device to carry out capillary electrophoresis, and data were collected using the GeneScan-500 LIZ dye Size Standard (Applied Biosystems, USA).
4
Genotyping of Parastagonospora nodorum
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Three expressed sequence tag (EST) derived SSR loci SNOD1, SNOD3, SNOD5, one minisatellite locus SNOD8 (Stukenbrock et al., 2005 (link)) and 16 newly developed SSR loci (Supplementary Table S1 ) were used for genotyping. The new SSR markers were designed by Dr. Patrick C. Brunner at ETH Zurich based on the reference genome SN15 and alignments with genome sequences of 164 global strains of P. nodorum (Pereira et al., 2019 (link)). PCR was carried out with M13 tailed (Schuelke, 2000 (link)) fluorescent labeled primers (Supplementary Table S1 ), PCR products were separated by capillary electrophoresis using an ABI3730 Gene Analyzer (Applied Biosciences) and a GeneScan 500 LIZ dye Size Standard from Applied Biosystems (Life Technologies), and the resulting data analyzed using Software: GeneMapper v.5 (Applied Biosystems).
5
DNA Fragment Analysis on ABI Prism 310
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Amplicons were separated on an ABI Prism® 310 Genetic Analyzer (Applied Biosystems® by life technologies, Darmstadt, Germany). PCR products were analyzed either undiluted or diluted in water after PCR. The standard dilution was either 1:5 or 1:10 (depending on the primer set). The (diluted) PCR product was denatured in formamide (Hi-Di™ Formamide, Applied Biosystems® by life technologies, Darmstadt, Germany) mixed with LIZ size standard (GeneScan™ 500 LIZ™ dye Size Standard, Applied Biosystems® by life technologies, Darmstadt, Germany) at 95°C for 5 min, followed by 10 min incubation on ice. Injection was performed at 15,000 V for 5 s. Electrophoresis was performed at 60°C, 15,000 V and 10 mA laser current. If the electrophoresis result was inadequate, the dilution and/or injection time were adjusted: If the signals reached the detection maximum, the dilution was increased. If the peaks were too small or absent, the injection time was increased to 10 s. If this did not improve the results, electrophoresis was repeated with less diluted or undiluted PCR product.
6
Microsatellite Genetic Diversity Analysis of A. antennatus
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From each specimen, DNA was extracted from the muscle tissue using the adjusted phenol-chloroform method proposed by Fernández et al. [28 (link)]. Genetic diversity was analysed at 10 microsatellite loci previously described for A. antennatus (Aa123, Aa138, Aa1444, Aa667, Aa681, Aa751, Aa956, Aa1061, Aa1195, and Aa818) [29 (link)], and amplified with three multiplex PCRs [10 (link)]. Resulting amplicons were analysed in an ABI PRISM 3730xl DNA analyser (Applied Biosystems, Foster City, CA, USA) at the Sequencing Unit of the University of Santiago de Compostela (Campus Lugo, Lugo, Spain), and were genotyped using GeneMapper software version 4.0 with GeneScan 500LIZ dye Size Standard (Applied Biosystems) as the internal standard. Genotype data will be available on request.
7
Primer Extension for RNA Analysis
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To perform primer extension, from 0.06 to 1 μg of total RNA from cell lines or Human XpressRef Universal Total RNA (Qiagen) complexed with 10 pmol of 5.8S Rev FAM primer and 2 pmol of 5S Rev Hex primer (Integrated DNA Technologies, HPLC purification) in a total volume of 3 μl was denatured at 92°C for 2.5 minutes and incubated at 59°C for 30 minutes for primer annealing followed by 5 minutes on ice. Then the mix containing water, GoScript Reaction Buffer, 5 mM MgCl2, dNTPs (0.5 mM each), 10 u of RNase inhibitor and 160 u of GoScript Reverse Transcriptase (GoScript Reverse Transcriptase kit Promega) was added to the sample to obtain a final volume of 10 μl and incubated at 42°C for 1 hour. This reaction is carried out in 0.2 ml tubes (Multiply-Pro cup PP Sarstedt) in a thermal cycler (T100 Bio-Rad). After primer extension, 1 μl of sample was added to 8.8 μl of Hi-Di Formamide (Applied Biosystems) and 0.2 μl of GeneScan 500 Liz dye size standard (Applied Biosystems). Samples were separated on 36 cm capillary array using POP-7 Polymer on an Abi 3730 Sequencer (Applied Biosystems) using DS-33 Matrix (Dye Set G5) for spectra calibration.
8
Microsatellite Analysis of Genomic DNA
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Genomic DNA from hair and tissue samples was extracted by Qiagen® DNeasy® Blood & Tissue Kits. In the case of blood samples, Geneaid™ Genomic DNA Mini Kits (Blood-Cultured Cell) were used. Twelve microsatellite loci were selected for the analyses because microsatellites are fast evolving markers suitable for analyses of recent population structure33 (link).
The PCRs were carried out in T100™ Thermal Cyclers from Bio-Rad using the Qiagen® Multiplex PCR kit according to the enclosed protocol34 . During reactions, 12 microsatellite loci were amplified with already published fluorescently labelled primers that were chosen based on cross-species amplification, including 5 previously used for WDE6 (link); for details see TableS1 in Supplementary Material. Fragmentation analyses was performed using a 3500 Genetic Analyzer (Applied Biosystems™) with the GeneScan™ 500 LIZ™ dye Size Standard.
The PCRs were carried out in T100™ Thermal Cyclers from Bio-Rad using the Qiagen® Multiplex PCR kit according to the enclosed protocol34 . During reactions, 12 microsatellite loci were amplified with already published fluorescently labelled primers that were chosen based on cross-species amplification, including 5 previously used for WDE6 (link); for details see Table
9
Microsatellite Genotyping Protocol
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PCR amplification was first assayed on a subsample of 6 individuals. PCR amplifications were performed using a Mastercycler (Eppendorf, Hamburg, Germany) thermocycler, in a 20 μL reaction mix containing 70 mM Tris-HCL, 2 mM MgCl2, 17 mM (NH4)2SO4, 10 mM β-mercaptoethanol, 0.05% (w/v) polyoxyethylene-ether W1, 0.2 mg/mL bovine serum albumin, 200 mM of each dNTP, 10 ng genomic DNA, 0.5 units of Taq DNA polymerase, and 0.2 μM each of reverse and forward primers. The PCR program used consisted of 5 min at 95 °C, followed by 37 cycles of 5 s at 95 °C, 10 s at 60 °C and 30 s at 72 °C. Amplicons were visualised under UV light by electrophoresis on a 3% (w/v) agarose gel stained with ethidium bromide. When a single, intense amplicon was obtained from all 6 plants, it was sequenced on both strands to confirm the presence of the expected microsatellite motif.
For genotyping, the DNA extracts of all plants were diluted 50-fold prior to genotyping with fluorescent dye-labelled markers (6-FAM, NED, VIC, PET). PCR products were assayed on an ABI 3730XL sequencer (Applied Biosystems, Foster City, CA, USA) using GeneScan 500 LIZ dye size standard (Applied Biosystems). Amplicon sizes were analyzed with the software Peak Scanner 1.0 (Applied Biosystems).
For genotyping, the DNA extracts of all plants were diluted 50-fold prior to genotyping with fluorescent dye-labelled markers (6-FAM, NED, VIC, PET). PCR products were assayed on an ABI 3730XL sequencer (Applied Biosystems, Foster City, CA, USA) using GeneScan 500 LIZ dye size standard (Applied Biosystems). Amplicon sizes were analyzed with the software Peak Scanner 1.0 (Applied Biosystems).
10
DNA Extraction and Microsatellite Genotyping
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We extracted DNA from blood and muscle tissue using the DNeasy blood and tissue Kit (Qiagen Inc., Valencia, CA, USA). For white-eared ground-sparrow, we amplified eight primers: Mme2, Mme7, Mme8, Asµ15, Asµ18, Escµ6, Gf01 and Gf0551 (link)–54 (link). For house wren, we amplified eight primers: ThPl-01, ThPl-14, ThPl-17, Ta-C6-7, Ta-B4-2, Ta-A5-2, Ta-A5-15 and Ta-C3(B)-255 (link),56 (link). Forward primer of each pair was 5’ fluorescently labelled. PCR thermal profiles and primer mixes are described in Supplementary Table S3 . We amplified all markers in a 12.5µL reaction containing 0.4 µM primer mix and 40 ng of template DNA. We used Multiplex Master Kit (Qiagen) for mixed primers and Top Taq Master Kit (Qiagen) for Gf01. We used Veriti™ thermal cycler (Applied Biosystems, Foster City, CA, USA) to perform the PCR reaction. Capillary electrophoresis was done in a 3500 Genetic Analyzer (Applied Biosystems) at Escuela de Biología, UCR, using Hi-Di™ Formamide and GeneScan™ 500 LIZ™ dye Size Standard (Applied Biosystems). We scored genotypes using GeneMarker 1.91 (SoftGenetics, State College, PA, USA).
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