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Random hexamers

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Random hexamers are short, single-stranded DNA sequences that consist of six random nucleotides. They can be used as primers for reverse transcription and amplification of RNA templates in various molecular biology applications.

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Lab products found in correlation

Qubit dsdna hs assay, by Thermo Fisher Scientific (4 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions) Phix control, by Illumina (2 mentions) Miseq barcodes, by Illumina (2 mentions) Indexing kit v2 set b, by Illumina (2 mentions) Superscript 3 rt enzyme, by Thermo Fisher Scientific (2 mentions) Miseq reagent kit, by Illumina (2 mentions) Allprep dna rna mini kit, by Qiagen (2 mentions) Ampure bead, by Beckman Coulter (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Dntp mix, by Thermo Fisher Scientific (1 mentions) Ssiv buffer, by Thermo Fisher Scientific (1 mentions) Ribolock rnase inhibitor, by Thermo Fisher Scientific (1 mentions) Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) 5 mm stainless steel bead, by Qiagen (1 mentions) Qiashreddertm columns, by Qiagen (1 mentions) Dntps, by Thermo Fisher Scientific (1 mentions) Mgcl2, by Merck Group (1 mentions)

7 protocols using random hexamers

1

Reverse Transcription Protocol for RNA Quantification

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Reverse transcription was performed on all samples using the following protocol. RNA (5 µL for samples or 1 µL standard RNA) was added to 100 ng random hexamers (Integrated DNA technologies, Coralville, IA, USA) and 1 µL dNTP mix (10 mM, Thermo Fisher Scientific, Waltham, MA, USA) in a final volume of 12 µL. This mixture was incubated at 65 °C for 5 min, followed by one-minute incubation on ice. Seven µL reaction mix containing 1× SSIV buffer (Invitrogen, Carlsbad, CA, USA), 5 M dithiothreitol (Invitrogen, Carlsbad, CA, USA), 40 U Ribolock RNase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), and 200 U SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was added and incubated at 23 °C for 10 min, 50 °C for 30 min, followed by incubation at 80 °C for 10 min. All reactions were stored at −70 °C until use.
Coertse J., Mortlock M., Grobbelaar A., Moolla N., Markotter W, & Weyer J. (2023). Development of a Pan-Filoviridae SYBR Green qPCR Assay for Biosurveillance Studies in Bats. Viruses, 15(4), 987.
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2

Profiling Immune Repertoire Dynamics

Cited 1 time
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Total RNA was extracted from cryopreserved PBMC at 7 different time-points (8, 11, 17, 34, 38, 46 and 108 wpi) using the AllPrep DNA/RNA mini kit (QIAGEN). Reverse transcription was carried out using Random Hexamers (Integrated DNA Technologies) and Superscript III RT enzyme (Invitrogen). Lineage specific primers bound leader regions of IGHV4-39 and IGLV3-21 and included the Illumina MiSeq barcodes to allow sequencing on the MiSeq (primers listed in the Key Resources Table). Samples from each time point were amplified as seven replicates for both the heavy and light chains, to ensure adequate coverage and to minimize PCR bias. PCR conditions were based on previous experiments (Scheepers et al., 2015 (link)) but modified by increasing the annealing temperature to 65°C and reducing cycles to 25. Nextera XT unique dual indexing combinations selected from Illumina Indexing Kit V2 Set B were added to the pooled MiSeq amplicon libraries. All products were checked on an Agilent Bioanalyser High Sensitivity DNA kit (Diagnostech) and Qubit dsDNA HS assay (ThermoFischer Scientific) and cleaned-up using 0.75X Ampure Beads (Beckman-Coulter). A final concentration of 4.5 pM denatured DNA library with 10% PhiX control (Illumina) was run on the Illumina MiSeq, using the MiSeq reagent kit (version 3) with 2 × 300 paired-end reads.
Scheepers C., Bekker V., Anthony C., Richardson S.I., Oosthuysen B., Moyo T., Kgagudi P., Kitchin D., Nonyane M., York T., Mielke D., Mabvakure B.M., Sheng Z., Lambson B.E., Ismail A., Garrett N.J., Karim S.S., Shapiro L., Williamson C., Morris L, & Moore P.L. (2020). Antibody Isotype Switching as a Mechanism to Counter HIV Neutralization Escape. Cell reports, 33(8), 108430.
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3

Comprehensive Tissue RNA and Protein Isolation

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RNA was isolated from the hippocampus, hypothalamus, colon, small intestine, and liver. Proteins were isolated from small intestine and colon tissues. Frozen samples were homogenized in TRIzol reagent (Life Technologies) on the TissueLyser II (QIAGEN) with one 5 mm stainless steel bead (QIAGEN) for 2 cycles of 2-min duration at 20 Hz. One-fifth volume chloroform was added to the tissue lysates to enable phase separation. The upper aqueous phase was homogenized by passage through QIAshredderTM columns (QIAGEN), and RNA was isolated from the aqueous phase using Qiagen RNeasy mini (Valencia, CA, USA) protocol for animal tissue. Total RNA yield was determined by absorbance at 260 nm, and purity was determined by the 260/280 nm ratio using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). RNA was treated with 1/5 concentration DNAse I (0.64 µL per 4 µg RNA; Life Technologies) in 25 mM MgCl2 (Sigma) for 30 min at 37 °C, then 10 min at 75 °C. DNAse-treated RNA was reverse transcribed to cDNA using SuperScript II Reverse Transcriptase (Life Technologies), dNTPs (Life Technologies), and random hexamers (Integrated DNA Technologies, Coralville) according to the manufacturer’s protocol.
Elizabeth de Sousa Rodrigues M., Bekhbat M., Houser M.C., Chang J., Walker D.I., Jones D.P., Oller do Nascimento C.M., Barnum C.J, & Tansey M.G. (2016). Chronic psychological stress and high-fat high-fructose diet disrupt metabolic and inflammatory gene networks in the brain, liver, and gut and promote behavioral deficits in mice. Brain, behavior, and immunity, 59, 158-172.
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4

Quantitative RT-PCR protocol for gene expression

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Samples frozen in 350 μL RLT Buffer (Qiagen) with 1 % 2-mercaptoethanol (Sigma-Aldrich) were thawed, and homogenized with a 21-gauge needle (Kendall). The mRNA was isolated using a phenol-chloroform extraction with MaXtract High Density Tubes (Qiagen). The subsequent mRNA purifications were carried out with a RNeasy Mini Kit (Qiagen) and RNase-Free DNase Set (Qiagen). The first-strand cDNA was then synthesized with 1 μg of mRNA from each sample using 100 ng/μL of random hexamers (Integrated DNA Technologies), Superscript II Reverse Transcriptase (Invitrogen), and RNaseOut Recombinant Ribonuclease Inhibitor (Invitrogen). The qRT-PCR reaction mixture was prepared with a SensiFAST SYBR Lo-ROX kit (Bioline), and primers from Integrated DNA Technologies that amplified either eef1a1l1 (Forward 5′-CCGTCTGCCACTTCAGGATGTGT-3′, Reverse 5′-TTGAGGACACCAGTCTCCACACGA-3′) or d4EGFP (Forward 5′-CGAGCAACTGAGGATCCCATTCTCT-3′, Reverse 5′-CACCCCGGTGAACAGCTCCT-3′). All qRT-PCR reactions were carried out on a Bio-Rad C1000 Touch Thermal Cycler CFX 96 Real-Time System with a polymerase activation step at 95 °C for 2 min, followed by 40 3-step cycles of 95 °C for 5 s, 58 °C for 10s, and 72 °C for 20 s, and finally a melting curve analysis cycle of 95 °C for 10 s, 65 °C for 5 s, and 95 °C for 50 s.
Krug RG I.I., Poshusta T.L., Skuster K.J., Berg M.R., Gardner S.L, & Clark K.J. (2014). A transgenic zebrafish model for monitoring glucocorticoid receptor activity. Genes, brain, and behavior, 13(5), 478-487.
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5

Transcriptomic Profiling of Atrx-Modulated OPCs

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cDNA was prepared using 100 ng RNA from forebrain (P14 AtrxFoxG1Cre), FANS-purified GFP+ OPC nuclei or from optic tracts (P20 AtrxSox10Cre) (RNeasy Mini kit; Qiagen Cat# 74104) and 1.5 μL of 100 ng/L of random hexamers (Integrated DNA Technologies Cat# 51-01-18-26) were diluted to a final volume of 12 μL with RNAse free water. Samples were heated at 65 °C for 10 min followed by addition of 4 µL first strand buffer (Thermo Fisher Scientific Cat# 18064014), 2 µL 100 mM DTT (Thermo Fisher Scientific Cat# 18064014), 0.8 μL 25 mM dNTPs, 0.5 μL RNaseOut (Thermo Fisher Scientific Cat# 10777019), 0.5 μL SuperScript II Reverse Transcriptase (Thermo Fisher Scientific Cat# 18064014) and 0.5 µL RNAse free water per sample. Samples were then incubated at 30 °C for 10 minutes and 42 °C for 45 min and stored at −20 °C. cDNA was amplified with iQ SYBR Green Master Mix (BioRad Cat# 1708884) using the standard curve Ct method of quantification. Experiments were performed on a Chromo-4 thermocycler (MJ Research/BioRad) and analyzed with Opticon Monitor 3 and GeneX (BioRad) software. Technical duplicates were completed for each sample. Conditions for amplification were as follows: 40 cycles of 95 °C for 10 s, 55–60 °C for 20 s, 72 °C for 30 s, and a final melting curve generated in increments of 0.5 °C per plate read. Primer sequences are listed in Supplementary Table 1.
Rowland M.E., Jiang Y., Shafiq S., Ghahramani A., Pena-Ortiz M.A., Dumeaux V, & Bérubé N.G. (2023). Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice. Nature Communications, 14, 7090.
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6

Profiling Immune Repertoire Dynamics

Cited 1 time
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Total RNA was extracted from cryopreserved PBMC at 7 different time-points (8, 11, 17, 34, 38, 46 and 108 wpi) using the AllPrep DNA/RNA mini kit (QIAGEN). Reverse transcription was carried out using Random Hexamers (Integrated DNA Technologies) and Superscript III RT enzyme (Invitrogen). Lineage specific primers bound leader regions of IGHV4-39 and IGLV3-21 and included the Illumina MiSeq barcodes to allow sequencing on the MiSeq (primers listed in the Key Resources Table). Samples from each time point were amplified as seven replicates for both the heavy and light chains, to ensure adequate coverage and to minimize PCR bias. PCR conditions were based on previous experiments (Scheepers et al., 2015 (link)) but modified by increasing the annealing temperature to 65°C and reducing cycles to 25. Nextera XT unique dual indexing combinations selected from Illumina Indexing Kit V2 Set B were added to the pooled MiSeq amplicon libraries. All products were checked on an Agilent Bioanalyser High Sensitivity DNA kit (Diagnostech) and Qubit dsDNA HS assay (ThermoFischer Scientific) and cleaned-up using 0.75X Ampure Beads (Beckman-Coulter). A final concentration of 4.5 pM denatured DNA library with 10% PhiX control (Illumina) was run on the Illumina MiSeq, using the MiSeq reagent kit (version 3) with 2 × 300 paired-end reads.
Scheepers C., Bekker V., Anthony C., Richardson S.I., Oosthuysen B., Moyo T., Kgagudi P., Kitchin D., Nonyane M., York T., Mielke D., Mabvakure B.M., Sheng Z., Lambson B.E., Ismail A., Garrett N.J., Karim S.S., Shapiro L., Williamson C., Morris L, & Moore P.L. (2020). Antibody Isotype Switching as a Mechanism to Counter HIV Neutralization Escape. Cell reports, 33(8), 108430.
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7

Quantitative Real-Time PCR of mRNA

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One μg of DNA-depleted samples of mRNA was incubated with random hexamers (Integrated DNA Technologies) at 65°C for 5 min and the mixtures transferred to ice. cDNA was prepared with a SuperScript III Reverse Transcriptase kit (Invitrogen) in total reaction volumes of 20 μL. SYBR Select Master Mix (Applied Biosystems) was used for RT-PCR. Briefly, 2× SYBR Select was mixed with primers (200 nM final concentration) (Table S3) and 18 μl were aliquoted per well. Finally, 20-μL cDNA reactions were diluted 1:5 and 2 μL added per well. A 7500 Fast real-time PCR system (Applied Biosystems) and software (v2.3) were used for cycle threshold quantification and relative gene expression analysis.
Simanek K.A., Taylor I.R., Richael E.K., Lasek-Nesselquist E., Bassler B.L, & Paczkowski J.E. (2022). The PqsE-RhlR Interaction Regulates RhlR DNA Binding to Control Virulence Factor Production in Pseudomonas aeruginosa. Microbiology Spectrum, 10(1), e02108-21.
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