Bigdye v1.1 kit

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, United States

The BigDye v1.1 kit is a DNA sequencing reagent kit produced by Thermo Fisher Scientific. It is used for the fluorescent dye-terminator DNA sequencing method.

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Lab products found in correlation

Expand long range pcr kit, by Roche (1 mentions) Abi 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions) Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions) Tempus blood rna tube, by Thermo Fisher Scientific (1 mentions) Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Monarch gel extraction kit, by New England Biolabs (1 mentions) Kapa express extract kit, by Roche (1 mentions)

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BigDye v1.1 (20 citations)

4 protocols using bigdye v1.1 kit

CADPS2 Deletion Breakpoint Cloning

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PCR primers for CADPS2 deletion breakpoint cloning were designed with Primer3 v4.0 as follows: forward 5′-GGCAGAGAGGATGACGTAG-3′; reverse 5′-CTGGATGGAGAAGAGCTGGA-3′. A long-range PCR was performed using the Expand-Long Range PCR kit according to the manufacturer's instruction (Roche Diagnostics) using 200 ng of genomic DNA from peripheral blood with the following PCR cycles (40): 92°C 2′, 92°C 10′′ 60°C 15′′ 68°C 14′, 68°C 7′.
PCR products were purified onto a Millipore PCR clean-up plate, cloned with the Original TA cloning kit (Life Technologies) in the pcDNA2.1 vector, and transformed into DH5α E. coli strains for white/blue screening. White colonies were grown and the plasmid DNA was purified and sequenced with universal M13 forward and reverse primers using the BigDye v1.1 kit (Life Technologies). Sequences were run onto the ABI 3730 automated sequencing machine, and electropherograms were analyzed with Chromas version 2.0.

Bonora E., Graziano C., Minopoli F., Bacchelli E., Magini P., Diquigiovanni C., Lomartire S., Bianco F., Vargiolu M., Parchi P., Marasco E., Mantovani V., Rampoldi L., Trudu M., Parmeggiani A., Battaglia A., Mazzone L., Tortora G., Maestrini E., Seri M, & Romeo G. (2014). Maternally inherited genetic variants of CADPS2 are present in Autism Spectrum Disorders and Intellectual Disability patients. EMBO Molecular Medicine, 6(6), 795-809.

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CADPS2 Gene Sequence Analysis

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PCR primers for human CADPS2 (NM_017954.10) were designed with Primer3 v4.0. Genomic DNA extracted from peripheral blood was amplified according to the following PCR conditions: 30 ng of DNA, 2.5 mM MgCl₂, 0.5 mM dNTPs, 0.5 μM primers, 5% DMSO in a final volume of 20 μl using the KAPA Fast Taq Polymerase Master mix (KAPA Biosystems, MA, USA). Forty cycles were carried out as follows: 95°C 1′, 95°C 15′′, 58°C 15′′, 72°C 20′′, with a final extension of 30′′ at 72°C. PCR products were purified onto Millipore PCR clean-up plates and directly sequenced on both strands using the BigDye v1.1 kit (Life Technologies). Electropherograms were visualized with Chromas version 2.0 and Sequencer version 4.7.
SNPs rs2074589 (chr7 g.122,078,414T > G) in exon 17 and rs20784589 (chr7 g.122,033,379A > G) in exon 22 were genotyped using the same PCR primers and conditions utilized for mutation screening.

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RNA Isolation and Sequencing Protocol

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RNA was isolated using the Tempus Spin RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) from peripheral blood taken in Tempus Blood RNA tubes (Thermo Fisher Scientific) according to the manufacturer’s recommendations. First-strand reverse transcription was carried out by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Reverse transcription PCRs (RT-PCRs) amplifying the variant-containing exons along with at least two adjacent exons were designed individually (list of cDNA primers is given in S2 Table). Amplification products were visualized on 1% agarose gel next to Hyper Ladder 1 kb DNA sizing standard (Bioline, London, UK) and subsequently sequenced by conventional Sanger sequencing method on ABI3130 Genetic Analyzer using the BigDye v.1.1 Kit (Thermo Fisher Scientific). Sequencing was done for the whole RT-PCR product without separation of the respective bands to compare peak intensities of normal and aberrantly spliced products. Where it was necessary to remove the interfering predominant normal alternative splice product, fragments of different sizes were cut out and cleaned from the gel by Monarch Gel Extraction Kit (New England Biolabs, Ipswich, MA) and the purified product was sequenced as above.

Bozsik A., Papp J., Grolmusz V.K., Patócs A., Oláh E, & Butz H. (2022). Reclassification of Five BRCA1/2 Variants with Unknown Significance Using Complex Functional Study. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 54(4), 970-984.

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Sanger Sequencing for MYD88 L265P

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We studied the remaining ten cases, which failed both WES and target panel sequencing, using Sanger sequencing for the hotspot MYD88 mutation L265P, and we were successful in seven cases. In brief, DNA was extracted from FFPE tissues using a KAPA Express Extract kit (Roche) upon microdissection. PCR amplification was performed using KAPA 2G Fast Readymix (Roche) and the primers flanking the L265 mutation hotspot of the MYD88 gene (forward: 5′-CCCACCATGGGGCAAGG-3′ and reverse: 5′-GGTGTAGTCGCAGACAGTGATGAA-3′). The PCR product was purified and sequenced using a BigDye v1.1 kit (ThermoFisher, Waltham, MA, USA).

Shi Z.F., Li K.K., Liu A.P., Chung N.Y., Wong S.C., Chen H., Woo P.Y., Chan D.T., Mao Y, & Ng H.K. (2024). The Molecular Landscape of Primary CNS Lymphomas (PCNSLs) in Children and Young Adults. Cancers, 16(9), 1740.

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