Prism 3100 genetic analyzer

Manufactured by Thermo Fisher Scientific

The PRISM 3100 Genetic Analyzer is an automated DNA sequencing system designed for high-throughput genetic analysis. It utilizes capillary electrophoresis technology to separate and detect fluorescently-labeled DNA fragments. The system is capable of analyzing a wide range of sample types, including plasmids, PCR products, and genomic DNA.

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Lab products found in correlation

Pmir report mirna expression reporter vector system, by Thermo Fisher Scientific (1 mentions) Bigdye terminator v1, by Thermo Fisher Scientific (1 mentions) Cycle sequencing kits, by Thermo Fisher Scientific (1 mentions) Bovine serum albumin (bsa), by New England Biolabs (1 mentions) Mastercycler gradient thermal cycler, by Eppendorf (1 mentions) Taq buffer, by New England Biolabs (1 mentions) Prism bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Genotyper software version 3, by Thermo Fisher Scientific (1 mentions) Pcr purification kit, by Thermo Fisher Scientific (1 mentions) Epitect bisulfite kit, by Qiagen (1 mentions)

Close spelling variants of this product

ABI PRISM 3100 Genetic Analyzer ABI Prism 3100 Genetic Analyzer sequencer ABI PRISM 3100 genetic analyzer DNA Sequencer ABI-Prism 3100 genetic analyzer system ABI PRISM® 3100 Avant Genetic Analyzer system PRISM 3100-Avant Genetic Analyzer ABI Prism 3100 DNA genetic analyzer ABI PRISM 3100 Avant Genetic Analyzer 3100 Genetic Analyzer Prism 3100

5 protocols using prism 3100 genetic analyzer

Luciferase Assay for miRNA Target Identification

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To identify the rno-miR-376a-binding site at the 3′-end of GRP78 mRNA, we generated a direct-match miRNA target site and cloned the insert into the multiple cloning site in the luciferase reporter vector from the pMIR-REPORT miRNA Expression Reporter Vector System (Applied Biosystems). The sense and antisense strands of the oligonucleotides were annealed by adding 2 µg of each oligonucleotide to 46 µL of annealing solution (100 mM potassium acetate, 30 mM HEPES-KOH, pH 7.4 and 2 mM magnesium acetate) and incubating at 90°C for 5 min and then at 37°C for 1 h. The annealed oligonucleotides were digested with HindIII and SpeI and ligated into the HindIII and SpeI restriction sites of pMIR-REPORT vector. The sequences of the inserts were confirmed by sequence analysis using a PRISM 3100 genetic analyzer (Applied Biosystems).
The following oligonucleotides were used in this studies: (1) miR-376a,
5′-aatgcactagtACGAGGATTTTCCTCTACGATaagcttaatgc-3′ and
5′-gcattaagcttATCGTAGAGGAAAATCCTCGTactagtgcatt-3′, and
(2) rno-miR-376a-binding site sequence at the 3′-end of GRP78 mRNA,
5′-aatgcactagtATGGTAGAAAAAAGTTCCTACaagcttaatgc-3′ and
5′-gcattaagcttGTAGGAACTTTTTTCTACCATactagtgcatt-3′

Iwamune M., Nakamura K., Kitahara Y, & Minegishi T. (2014). MicroRNA-376a Regulates 78-Kilodalton Glucose-Regulated Protein Expression in Rat Granulosa Cells. PLoS ONE, 9(10), e108997.

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Sequencing of IDH1/2 and BRAF

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PCR products were purified using ExoSap-IT (Affymetrix/USB, Santa Clara, CA, USA). Thereafter, cycle sequencing was carried out using BigDye^® Terminator v1.1 (for IDH1/2) or v3.1 (for BRAF) Cycle Sequencing Kits (Applied Biosystems). Following purification, the electrophoresis and analyses were conducted using a PRISM^® 3100 Genetic Analyzer (Applied Biosystems). Raw data were analyzed using the phred/phrap/consed package (http://www.phrap.org/consed/consed.html#howToGet) for sequence determination.

Hatae R., Hata N., Yoshimoto K., Kuga D., Akagi Y., Murata H., Suzuki S.O., Mizoguchi M, & Iihara K. (2016). Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data. PLoS ONE, 11(8), e0160489.

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Amplification and Genotyping of Symbiodinium fitti Microsatellites

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Symbiodinium “fitti” DNA was amplified with primers given by Pinzón, Devlin‐Durante, Weber, Baums, and LaJeunesse (2011) (n = 10 loci). Three additional loci for S. “fitti” were developed de novo (Table S1). Alleles were fluorescently visualized and sized with internal standards on a PRISM 3100 Genetic Analyzer (Applied Biosystems). Briefly, 25–50 ng of template DNA was added to polymerase chain reactions (PCRs) containing 1 × Standard Taq Buffer (New England Biolabs), 2.5 mm MgCl (New England Biolabs), 0.5 mg/ml bovine serum albumin (New England Biolabs) and 0.75 U of Taq (0.325 U for primers 31, 32 and 41; New England Biolabs). Each primer was added at 200 nm to reactions involving loci A3Sym_01, 18, 27 and 28, whereas a primer concentration of 93 nm was added to reactions for primers 31, 32 and 41. Concentrations of 50 nm of the tailed forward primer (see Table S1), 150 nm of the reverse primer and 75 nm of the dye‐labelled T‐oligonucleotide were used for amplifying loci A3Sym_01, 02, 03, 07, 08, 09 and 48. All loci were amplified using the following thermal cycle profile: 94°C for 2 min (one cycle); 94°C for 15 s, primer‐specific annealing temperature (Table S1) for 15 s and 72°C for 30 s (31 cycles); and 72°C for 30 min on a Mastercycler gradient thermal cycler (Eppendorf). Table S2 provides details of microsatellite genotype allele calls.

Durante M.K., Baums I.B., Williams D.E., Vohsen S, & Kemp D.W. (2019). What drives phenotypic divergence among coral clonemates of Acropora palmata?. Molecular Ecology, 28(13), 3208-3224.

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Genotyping the GSTP1 Gene Polymorphism

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Genomic DNA was extracted from 3 ml peripheral blood using proteinase K treatment and the standard phenol-chloroform extraction procedure^{46 (link)}. The details of primer sequences (Table S2) and about the PCR reaction are given in the Supplementary section.
The genetic polymorphism of GSTP1 in exon 5 (rs1695, Ile/Ile, Ile/Val, Val/Val genotypes) was identified using the Alw26I restriction enzyme^{47 (link)}. A 433 bp fragment of GSTP1 gene was amplified and the presence of Alw26I restriction site yielded 328 and 105 bp fragments, respectively (GSTP1 Ile/Ile). The presence of rs1695 (313 A/G) creates another restriction site within the 328 bp fragment which when digested by Alw26I, yielded two fragments of 222 and 106 bp (GSTP1 Val/Val) (Fig. 1A,B).
To validate the genotype data generated by PCR-RFLP for rs1695, a subset of samples (100 in each) were resequenced for the GSTP1 gene using the same primer pairs that were used for PCR during the RFLP assay. The sequencing reactions were performed by conventional Sanger sequencing method using an ABI PRISM 3100 Genetic Analyzer and the genotypes were determined from the electropherograms using Seqscape v.2.4 (Applied Biosystems) (Fig. 1C).

Yadav P., Banerjee A., Boruah N., Singh C.S., Chatterjee P., Mukherjee S., Dakhar H., Nongrum H.B., Bhattacharjee A, & Chatterjee A. (2020). Glutathione S-transferasesP1 AA (105Ile) allele increases oral cancer risk, interacts strongly with c-Jun Kinase and weakly detoxifies areca-nut metabolites. Scientific Reports, 10, 6032.

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Bisulfite DNA Sequencing Protocol

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We confirmed the MBD array data using bisulfite direct sequencing. DNA samples (1 μg) were bisulfite-converted using an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s instructions. The bisulfite-converted DNA was then amplified by PCR with primers for discrimination of the methylated and unmethylated CpG sites. The sequences of PCR primers are presented in Table 1. PCR reaction solutions contained 10 ng genomic DNA, 10 pM primers, 0.25 mM dNTPs, 1.5 mM MgCl₂, 1 X buffer, and 0.25 U Taq polymerase per 20 μL of total reaction volume. PCR conditions included predenaturation at 95°C for 10 min, 35 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 40 sec, and final extension at 72°C for 10 min. After PCR amplification, PCR products were purified using a PCR purification kit (Bioneer, Daejeon, Korea) and sequenced using a PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster city, USA) according to manufacturer’s instructions. Sequencing products were analyzed using a PRISM 3100 Genetic Analyzer (Applied Biosystems), and electropherogram traces were interpreted using Genescan software version 3.7 (Applied Biosystems). Corresponding genotypes were assigned using Genotyper software version 3.7 (Applied Biosystems).

Lim J.H., Lee D.E., Park S.Y., Kim D.J., Ahn H.K., Han Y.J., Kim M.Y, & Ryu H.M. (2014). Disease specific characteristics of fetal epigenetic markers for non-invasive prenatal testing of trisomy 21. BMC Medical Genomics, 7, 1.

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