E gel sizeselect gels

Manufactured by Thermo Fisher Scientific

Sourced in United States

The E-Gel SizeSelect Gels are precast agarose gels designed for quick and efficient size selection of DNA fragments. The gels provide a simple and reliable method for isolating specific DNA bands from complex mixtures.

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Lab products found in correlation

9 protocols using e gel sizeselect gels

Genomic Deletion Mapping Using PCR and Sanger Sequencing

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Patient blood DNA was extracted using Promega Maxwell 16 Blood DNA Purification Kits (Promega). Primers were designed adjacent to the estimated deleted region boundaries, and a Qiagen Taq PCR Core Kit was used to amplify the deletion boundaries. DNA fragments were gel‐purified using E‐Gel SizeSelect Gels (Life Technologies). DNA sequencing was performed by PCR using a Qiagen Taq PCR Core Kit (Qiagen) according to the manufacturer's specifications, followed by bidirectional sequencing using the Big Dye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's specifications and run on an ABI 3130xl or 3730 Genetic Analyzer (Applied Biosystems). Sanger sequencing was conducted at the CCR Genomics Core at the National Cancer Institute, NIH, Bethesda, MD. Forward and reverse sequences were evaluated using Sequencher 5.0.1 (Genecodes). All deletion breakpoint coordinates and Alu locations are based on the GRCh37/hg19 genome build.

Vocke C.D., Ricketts C.J., Schmidt L.S., Ball M.W., Middelton L.A., Zbar B, & Linehan W.M. (2021). Comprehensive characterization of Alu‐mediated breakpoints in germline VHL gene deletions and rearrangements in patients from 71 VHL families. Human Mutation, 42(5), 520-529.

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Sequencing Tumor-Specific Genetic Variations

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DNA from three individual tumors and peripheral blood leucocytes were evaluated Primers designed to amplify exon 2 of IDH2 and all three coding exons of the VHL gene were used to amplify specific DNA fragments using the Qiagen Taq PCR Core Kit (Germantown, MD). DNA fragments were gel purified using E-Gel SizeSelect Gels (Life Technologies, Grand Island, NY) and bi-directionally sequenced using Big Dye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems) and processed on an ABI 3130xl Genetic Analyzer.

Merino M.J., Ricketts C.J., Moreno V., Yang Y., Fan T.W., Lane A.N., Meltzer P.S., Vocke C.D., Crooks D.R, & Linehan W.M. (2021). Multifocal renal cell carcinomas with somatic IDH2 mutation: report of a previously undescribed neoplasm. The American journal of surgical pathology, 45(1), 137-142.

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Aneuploidy Analysis by NGS and aCGH

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The biopsied cells were whole genome amplified (WGA) using the Sureplex DNA Amplification System (Bluegnome) according to the manufacturer’s protocol. The WGA products were then processed for aneuploidy analysis by NGS or aCGH. Testing by aCGH was processed using Bluegnome 24sure V3 protocol (Illumina, Inc.) according to the manufacturer’s protocol. WGA products were fluorescently labeled with Cy3 and Cy5 dyes and random primers and subsequently were prepared to be hybridized to 24sure V3 array slides. Aneuploidy data analysis was performed using BlueFuse Multi Software. Testing by NGS was processed using Ion Torrent PGM (Ion Torrent) technology. Libraries were prepared by fragmenting WGA products with DNA concentrations of 100 ng using Ion Xpress Plus Fragment Library Kit (Life Technologies). Library fragments were selected at 200 bp using E-Gel SizeSelect Gels (Life Technologies) and then were normalized to 100 pM using an Ion Library Equalizer kit (Life Technologies). Libraries were subsequently pooled together into a mastermix and clonal amplified on the Ion One Touch 2 system. The template was then loaded into a 316 V2 chip (Life Technologies) and sequenced at 200 bp. Aneuploidy data analysis was performed using Ion Reporter software, using Low-Coverage Whole-Gnome workflow.

Coates A., Bankowski B.J., Kung A., Griffin D.K, & Munne S. (2016). Differences in pregnancy outcomes in donor egg frozen embryo transfer (FET) cycles following preimplantation genetic screening (PGS): a single center retrospective study. Journal of Assisted Reproduction and Genetics, 34(1), 71-78.

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DNA Extraction and Sequencing Protocol

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Patient blood DNA was extracted using Promega Maxwell 16 Blood DNA Purification Kits (Promega, WI, USA). Primers were designed adjacent to the estimated deleted region boundaries, and a Qiagen Taq PCR Core Kit (Germantown, MD) was used to amplify the deletion boundaries. DNA fragments were gel purified using E-Gel SizeSelect Gels (Life Technologies, Grand Island, NY). DNA sequencing was performed by PCR using a Qiagen Taq PCR Core Kit (Qiagen, MD, USA) according to the manufacturer’s specifications, followed by bidirectional sequencing using the Big Dye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) according to the manufacturer’s specifications and run on an ABI 3130xl or 3730 Genetic Analyzer (Applied Biosystems, CA, USA). Sanger sequencing was conducted at the CCR Genomics Core at the National Cancer Institute, NIH, Bethesda, MD 20892. Forward and reverse sequences were evaluated using Sequencher 5.0.1 (Genecodes, MI, USA). All deletion breakpoint coordinates and Alu locations are based on the GRCh37/hg19 genome build.

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Deletion Boundary Amplification Protocol

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Primers were designed adjacent to the estimated deleted region boundaries, and a Qiagen Taq PCR Core Kit (Germantown, MD) was used to amplify the deletion boundaries. DNA fragments were gel purified using E-Gel SizeSelect Gels (Life Technologies, Grand Island, NY).

Vocke C.D., Ricketts C.J., Merino M.J., Srinivasan R., Metwalli A.R., Middelton L.A., Peterson J., Yang Y, & Linehan W.M. (2017). Comprehensive genomic and phenotypic characterization of germline FH deletion in hereditary leiomyomatosis and renal cell carcinoma. Genes, chromosomes & cancer, 56(6), 484-492.

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RNA-seq Library Preparation and Sequencing

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RNA-seq data are deposited in GEO under the accession number GSE74979. For each sample, the non-ribosomal fraction of 3 μg of total RNA was isolated using a Ribo-Zero rRNA removal Kit (Epicentre, Madison, Wisconsin, USA). Ribo-Zero-treated RNAs were used to generate barcoded cDNA libraries using the TruSeq RNA Sample Preparation kit (Illumina), with the following additions. Size selections were performed before and after cDNA amplification on an E-gel Safe Imager (Invitrogen) using 2% E-gel SizeSelect gels (Invitrogen). The cDNA fraction of 300 bp in size (including adapters) was isolated and purified. Indexed libraries were pooled and sequenced (paired-end 50 or 100 bp reads) on the Illumina HiSEQ platform to a depth of 41–70 million reads per sample (QB3 Vincent J. Coates Genomics Sequencing Laboratory). After removal of PCR duplicates and repeats, there were 21–26 million uniquely mapping paired-end reads (37–61%).

de Bruin R.G., Shiue L., Prins J., de Boer H.C., Singh A., Fagg W.S., van Gils J.M., Duijs J.M., Katzman S., Kraaijeveld A.O., Böhringer S., Leung W.Y., Kielbasa S.M., Donahue J.P., van der Zande P.H., Sijbom R., van Alem C.M., Bot I., van Kooten C., Jukema J.W., Van Esch H., Rabelink T.J., Kazan H., Biessen E.A., Ares Jr. M., van Zonneveld A.J, & van der Veer E.P. (2016). Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression. Nature Communications, 7, 10846.

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Transcriptome Profiling via RNA-seq

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RNA sequencing analysis (RNA-seq) was performed, as previously described.^{6 (link)} Briefly, ribosomal RNA was removed using Ribo-Zero rRNA Removal Kit (Illumina). Sequence libraries for mRNA were generated using TruSeq RNA Library Prep Kit (Illumina). The cDNA fragments, ∼300 bp in size, were isolated and purified using 2% E-gel SizeSelect gels (Invitrogen). The libraries were then pair-end sequenced on an Illumina HiSEQ platform to a depth of 41–70 million reads per sample. The sequencing results were mapped using Tophat2 software to GRCh37/UCSC-hg19 genome assembly with the gene model provided by ‘hg19 UCSC Known Genes’.^{13 (link)} Guidance cues with non-zero counts were included for subsequent analyses.

Zhang H., Prins J., Vreeken D., Florijn B.W., de Bruin R.G., van Hengel O.R., van Essen M.F., Duijs J.M., Van Esch H., van der Veer E.P., van Zonneveld A.J, & van Gils J.M. (2020). Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking. Innate Immunity, 27(2), 118-132.

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mtDNA Sequencing Library Preparation

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Following confirmation of mtDNA amplification, 100 ng of the amplified mtDNA was used to generate a sequencing library with NEBNext® Fast DNA Fragmentation & Library Prep Set for Ion Torrent ™ (NEB), strictly following the manufacturer’s recommendations. The steps for library construction were as follows: (i) fragmentation of mtDNA to 100–400 bp; (ii) adapter ligation and, if sequencing a large number of specimens in one run, differentiation of specimens using an Ion Xpress Barcode Adapters 1–16 Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.); (iii) purification of adapter ligated DNA using Agencourt AMPure XP (Beckman); (iv) selection for 200-bp fragments using E-Gel® SizeSelect ™ gels (Thermo Fisher Scientific); (v) amplification by PCR; and (vi) purification of the amplified library using Agencourt AMPure XP.
The concentration of the completed library was measured using a Agilent 2100 bioanalyzer and a High Sensitivity DNA Kit (Agilent, Santa Clara, CA, U.S.A.).

Yao Y., Nishimura M., Murayama K., Kuranobu N., Tojo S., Beppu M., Ishige T., Itoga S., Tsuchida S., Mori M., Takayanagi M., Yokoyama M., Yamagata K., Kishita Y., Okazaki Y., Nomura F., Matsushita K, & Tanaka T. (2019). A simple method for sequencing the whole human mitochondrial genome directly from samples and its application to genetic testing. Scientific Reports, 9, 17411.

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Multiplexed NGS Sequencing Protocol

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Before next-generation sequencing, product size selection was performed using 2 % E-Gel SizeSelect Gels (Thermo Scientific). For expression and normalization reads, 6 cDNA samples and 6 gDNA samples were multiplexed in one lane and sequenced with 65-bp single reads using a HiSeq2500 (Illumina) yielding 210 × 10⁶ reads. For integration mapping, triplicate indexed PCR samples were pooled and sequenced with 75-bp paired-end reads using a MiSeq (Illumina) yielding 24 × 10⁶ reads. Reads were mapped to Drosophila melanogaster genome release dm3 using Bowtie.

Brueckner L., van Arensbergen J., Akhtar W., Pagie L, & van Steensel B. (2016). High-throughput assessment of context-dependent effects of chromatin proteins. Epigenetics & Chromatin, 9, 43.

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