Ceqtm 8000 genetic analysis system

Manufactured by Beckman Coulter

Sourced in United Kingdom

The CEQTM 8000 Genetic Analysis System is a capillary electrophoresis instrument designed for DNA fragment analysis. It utilizes fluorescence detection to separate and analyze DNA samples. The system is capable of performing various genetic analysis applications, including DNA sequencing, microsatellite analysis, and fragment sizing.

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Lab products found in correlation

Gexp starter kit, by Beckman Coulter (2 mentions) G storm gs1 thermal cycler, by Beckman Coulter (2 mentions) Thermo start taq dna polymerase, by Thermo Fisher Scientific (1 mentions) Genemapper v4, by Thermo Fisher Scientific (1 mentions) Abi 3730 sequencer, by Thermo Fisher Scientific (1 mentions) Ultraclean tissue cells dna isolation kit, by Qiagen (1 mentions) Ultraclean bloodspin kit, by Qiagen (1 mentions) Gs500liz size standard, by Thermo Fisher Scientific (1 mentions) Genomelab dtcs quick start kit, by Beckman Coulter (1 mentions) Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions) Cleanseq system, by Beckman Coulter (1 mentions) Faststart taq dna polymerase, by Roche (1 mentions)

6 protocols using ceqtm 8000 genetic analysis system

Multiplex Assays for Natriuretic Peptide Genes

Cited 3 times

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Customized GeXP multiplex assays were designed to genes that included the natriuretic peptide system (see Supplementary Table S2). Target-specific reverse transcription (using 100 ng RNA as template), and PCR amplification were performed as we described previously [49 (link),55 (link),56 (link)], and in accordance with manufacturer’s instructions (Beckman Coulter, High Wycombe, UK). In brief, a master mix was prepared for reverse transcription as detailed in the GeXP starter kit (Beckman Coulter, High Wycombe, UK) and performed using a G-storm GS1 thermal cycler (GStorm Ltd., Somerset, UK), using the following protocol: 48 °C 1 min; 42 °C 60 min; and 95 °C 5 min. From this an aliquot of each reverse transcriptase reaction was added to PCR master mix, consisting of GenomeLab kit PCR reaction mix and Thermoscientific Thermo-Start Taq DNA polymerase. PCR reactions were performed using G-storm GS1 thermal cycler with a 95 °C activation step for 10 min, followed by 35 cycles of 94 °C 30 s; 55 °C 30 s; 70 °C 60 s. Products were separated and quantified using CEQTM 8000 Genetic Analysis System, and GenomeLab Fragment Analysis software (eXpress Analysis Version 1.0.25, Beckman Coulter, High Wycombe, UK).

Lessey A.J., Mirczuk S.M., Chand A.N., Kurrasch D.M., Korbonits M., Niessen S.J., McArdle C.A., McGonnell I.M, & Fowkes R.C. (2023). Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development. International Journal of Molecular Sciences, 24(16), 12921.

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Multiplex MLPA for Hereditary Cancer

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The samples were screened for large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA). MLPA is a method for copy number detection by the multiplex PCR method. Small (50–70 nt) sequences are targeted, enabling MLPA to identify single exon aberrations. The samples were ligated and amplified using the SALSA MLPA P003 MLH1/MSH2 probe mix version B2 according to the protocol manufacturer's recommendation (MRC-Holland). The P003 MLH1/MSH2 probe mix version 2 contains probes for each of the 19 exons of the MLH1 gene and for each of the 16 exons of the MSH2 gene. Also, 2 probes are included for the most 3′ exon of EPCAM, a gene located just upstream of the MSH2 gene. Deletions of the most 3′ exon of the EPCAM gene can result in silencing of the MSH2 gene. In addition, the P003 MLH1/MSH2 probe mix also covers 7 genes in the CDKN2A-9p21 region + PAX5 (9p13) DOCK8 (9p24.3), and GLDC (9p21.1). The samples were analyzed on a CEQTM 8000 GeneticAnalysis System (Beckman Coulter Inc.). Data normalization and analysis were performed with GeneMarker Software version 1.75 (SoftGenetics) using standard parameters.

Andersson U., Wibom C., Cederquist K., Aradottir S., Borg Å., Armstrong G.N., Shete S., Lau C.C., Bainbridge M.N., Claus E.B., Barnholtz-Sloan J., Lai R., Il'yasova D., Houlston R.S., Schildkraut J., Bernstein J.L., Olson S.H., Jenkins R.B., Lachance D.H., Wrensch M., Davis F.G., Merrell R., Johansen C., Sadetzki S., Bondy M.L., Melin B.S., Adatto P., Morice F., Payen S., McQuinn L., McGaha R., Guerra S., Paith L., Roth K., Zeng D., Zhang H., Yung A., Aldape K., Gilbert M., Weinberger J., Colman H., Conrad C., de Groot J., Forman A., Groves M., Levin V., Loghin M., Puduvalli V., Sawaya R., Heimberger A., Lang F., Levine N., Tolentino L., Saunders K., Thach T.T., Iacono D.D., Sloan A., Gerson S., Selman W., Bambakidis N., Hart D., Miller J., Hoffer A., Cohen M., Rogers L., Nock C.J., Wolinsky Y., Devine K., Fulop J., Barrett W., Shimmel K., Ostrom Q., Barnett G., Rosenfeld S., Vogelbaum M., Weil R., Ahluwalia M., Peereboom D., Staugaitis S., Schilero C., Brewer C., Smolenski K., McGraw M., Naska T., Rosenfeld S., Ram Z., Blumenthal D.T., Bokstein F., Umansky F., Zaaroor M., Cohen A., Tzuk-Shina T., Voldby B., Laursen R., Andersen C., Brennum J., Henriksen M.B., Marzouk M., Davis M.E., Boland E., Smith M., Eze O., Way M., Lada P., Miedzianowski N., Frechette M., Paleologos N., Byström G., Svedberg E., Huggert S., Kimdal M., Sandström M., Brännström N., Hayat A., Tihan T., Zheng S., Berger M., Butowski N., Chang S., Clarke J., Prados M., Rice T., Sison J., Kivett V., Duo X., Hansen H., Hsuang G., Lamela R., Ramos C., Patoka J., Wagenman K., Zhou M., Klein A., McGee N., Pfefferle J., Wilson C., Morris P., Hughes M., Britt-Williams M., Foft J., Madsen J., Polony C., McCarthy B., Zahora C., Villano J., Engelhard H., Borg A., Chanock S.K., Collins P., Elston R., Kleihues P., Kruchko C., Petersen G., Plon S., Thompson P., Johansen C., Sadetzki S., Melin B., Bondy M.L., Lau C.C., Scheurer M.E., Armstrong G.N., Liu Y., Shete S., Yu R.K., Aldape K.D., Gilbert M.R., Weinberg J., Houlston R.S., Hosking F.J., Robertson L., Papaemmanuil E., Claus E.B., Claus E.B., Barnholtz-Sloan J., Sloan A.E., Barnett G., Devine K., Wolinsky Y., Lai R., McKean-Cowdin R., Il'yasova D., Schildkraut J., Sadetzki S., Yechezkel G.H., Bruchim R.B., Aslanov L., Sadetzki S., Johansen C., Kosteljanetz M., Broholm H., Bernstein J.L., Olson S.H., Schubert E., DeAngelis L., Jenkins R.B., Yang P., Rynearson A., Andersson U., Wibom C., Henriksson R., Melin B.S., Cederquist K., Aradottir S., Borg Å., Merrell R., Lada P., Wrensch M., Wiencke J., Wiemels J., McCoy L., McCarthy B.J, & Davis F.G. (2014). Germline rearrangements in families with strong family history of glioma and malignant melanoma, colon, and breast cancer. Neuro-Oncology, 16(10), 1333-1340.

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Multiplex GeXP Assay for Somatotropic Markers

Cited 3 times

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Customised GeXP multiplex assays were designed to genes that included the natriuretic peptide system as well as somatotrope markers (see Supplementary Table S1). Target-specific reverse transcription (using 100 ng RNA as template), and PCR amplification were performed as we described previously [15 (link),43 (link),44 (link)], and in accordance with manufacturer’s instructions (Beckman Coulter, High Wycombe, UK). In brief, a master mix was prepared for reverse transcription as detailed in the GeXP starter kit (Beckman Coulter, High Wycombe, UK) and performed using a G-storm GS1 thermal cycler (Agilegene Technologies Ltd., Somerton, Somerset, UK), using the following protocol: 48 °C 1 min; 42 °C 60 min; and 95 °C 5 min. From this an aliquot of each reverse transcriptase reaction was added to PCR master mix, consisting of GenomeLab kit PCR reaction mix and Thermo Scientific Thermo-Start Taq DNA polymerase. PCR reactions were performed using G-storm GS1 thermal cycler with a 95 °C activation step for 10 min, followed by 35 cycles of 94 °C 30 s; 55 °C 30 s; 70 °C 60 s. Products were separated and quantified using CEQTM 8000 Genetic Analysis System, and GenomeLab Fragment Analysis software (Beckman Coulter, High Wycombe, UK).

Mirczuk S.M., Scudder C.J., Read J.E., Crossley V.J., Regan J.T., Richardson K.M., Simbi B., McArdle C.A., Church D.B., Fenn J., Kenny P.J., Volk H.A., Wheeler-Jones C.P., Korbonits M., Niessen S.J., McGonnell I.M, & Fowkes R.C. (2021). Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours. International Journal of Molecular Sciences, 22(3), 1076.

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Microsatellite Genotyping from Diverse Samples

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DNA was extracted from blood and tissue samples either with the standard phenol–chloroform method [50 ], or using the UltraClean® BloodSpin™ Kit or UltraClean® Tissue & Cells DNA Isolation Kit (MoBio Laboratories), and from feathers using the method described in Rönkä et al. [34 (link)]. Individuals were genotyped for 12 microsatellite loci, which were amplified in 10 µl volumes containing 20–100 ng of template DNA, 0.1 µM of each primer, 0.8–1 mM MgCl₂, 0.2 mM of dNTPs, 1 µl of 10 × PCR-Buffer and 0.l U of DNA-polymerase (Biotools). The amplification profile was 94 °C for 1 min followed by 35 cycles of 94 °C for 30 s, 52–58 °C for 45 s (see Additional file 1: Table S1), 72 °C for 45 s and 72 °C for 10 min for final extension. The PCR reactions were run with ABI 3730 sequencer using GS500-Liz size standard (Applied Biosystems) and the loci were scored with GeneMapper v. 4.0. (Applied Biosystems), except for the Swedish samples, which were scored with CEQ^TM8000 Genetic Analysis System (Beckman Coulter) using the Fragment Analysis Module v. 8.0.52. Due to possible differences between the allele sizes defined by the two sequencers, samples were calibrated by genotyping five Swedish individuals with both sequencers. Genotyping error rate was calculated by amplifying most individuals twice. If differences were found between the two runs, samples were genotyped twice more.

Rönkä N., Pakanen V.M., Pauliny A., Thomson R.L., Nuotio K., Pehlak H., Thorup O., Lehikoinen P., Rönkä A., Blomqvist D., Koivula K, & Kvist L. (2021). Genetic differentiation in an endangered and strongly philopatric, migrant shorebird. BMC Ecology and Evolution, 21, 125.

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Targeted Sequencing of ETV6, CDKN1B, and TP53

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Exons 1-8 of ETV6 (ENSG00000139083), exons 1-2 of CDKN1B (ENSG00000111276), and exons 2-11 of TP53 (ENSG00000141510) were PCR-amplified from genomic DNA using FastStart Taq DNA Polymerase (Roche, Mannheim, Germany). After purification with the magnetic bead-based CleanSEQ® system (Beckman Coulter, Krefeld, Germany), PCR fragments were sequenced in both directions using the GenomeLab™ DTCS Quick Start Kit and CEQTM 8000 Genetic Analysis System (Beckman Coulter, Krefeld, Germany). Cloning of PCR products was performed in patients with complex mutations to describe the mutations properly using the TOPO TA Cloning® Kit (Invitrogen, Karlsruhe, Germany). All mutations were described according to the nomenclature for the description of sequence variations of the Human Genome Variation Society (HGVS, http://www.hgvs.org/).

Feurstein S., Rücker F.G., Bullinger L., Hofmann W., Manukjan G., Göhring G., Lehmann U., Heuser M., Ganser A., Döhner K., Schlegelberger B, & Steinemann D. (2014). Haploinsufficiency of ETV6 and CDKN1B in patients with acute myeloid leukemia and complex karyotype. BMC Genomics, 15(1), 784.

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SSR Markers for Indonesian Germplasm

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After quantification, the DNA samples were diluted to approximately 10 ng/μL. A total of 11 codominant markers developed by Molosiwa et al. (2015) were used to assess the variation of Indonesian materials (Table S2). The allele sizes were scored after the fragments were separated using the CEQ TM 8000 Genetic Analysis System (Beckman Coulter) with a 400bp internal standard. Visual investigation of the allele pattern combined with the automated scoring software were used to interpret the capillary electrophoresis results.
With the inclusion of data from samples reported by Molosiwa et al (2015) , the allelic sizes of 11 SSR markers were scored from a total of 170 accessions.

Redjeki E.S., Ho W.K., Shah N., Molosiwa O.O., Ardiarini N.R., K.u.s.w.a.n.t.o, & Mayes S. (2020). Understanding the genetic relationships between Indonesian bambara groundnut landraces and investigating their origins. Genome, 63(6).

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