Megabace 1000 automated sequencer

Manufactured by GE Healthcare

Sourced in China, United Kingdom

The MegaBACE 1000 is an automated DNA sequencing system designed for high-throughput genetic analysis. It utilizes capillary electrophoresis technology to perform DNA sequencing tasks efficiently.

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Lab products found in correlation

Multigene gradient thermal cycler, by Labnet (2 mentions) Abi 3700 sequencer, by Thermo Fisher Scientific (1 mentions) Dyenamic et dye terminator cycle sequencing kit, by GE Healthcare (1 mentions) Dyenamic et terminator cycle sequencing kit, by GE Healthcare (1 mentions) High pure pcr product purification kit, by Roche (1 mentions) Lasergene software, by DNASTAR (1 mentions) Multiplex master mix, by Qiagen (1 mentions) Fragment profiler 1, by Cytiva (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions)

4 protocols using megabace 1000 automated sequencer

Molecular Phylogenetic Protocol for Reptiles

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DNA was extracted from scales, shed skin, liver or muscle tissues using the
phenol:chloroform method following specific protocols for each tissue [40 ,41 ]. PCRs were performed using standard
protocols [41 ] for 11
genes, including four mitochondrial (12S, 16S, cox1,
cytb) and seven nuclear (amel,
bdnf, c-mos, jun,
hoxa13, nt3, rag1). The
sequences for each pair of primers and their respective references are provided
as supporting information (S8 Table). PCRs were purified with shrimp
alkaline phosphatase and exonuclease I (GE Healthcare, Piscataway, NJ).
Sequences were generated in Brazil at the Laboratório de Biologia Genômica e
Molecular, Pontifícia Universidade Católica do Rio Grande do Sul (Porto Alegre,
Rio Grande do Sul) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit in
a MegaBACE 1000 automated sequencer (GE Healthcare); and in China at Laboratory
for Conservation and Utilization of Bio-resources, Yunnan University (Kunming,
Yunnan) using BigDye Terminator cycle sequencing kit in an ABI 3700 sequencer
(Applied Biosystems, Foster City, CA). Both strands were sequenced for all
fragments and sequences were edited and assembled using Geneious 5.5 (http://www.geneious.com) [42 (link)].

Zaher H., Murphy R.W., Arredondo J.C., Graboski R., Machado-Filho P.R., Mahlow K., Montingelli G.G., Quadros A.B., Orlov N.L., Wilkinson M., Zhang Y.P, & Grazziotin F.G. (2019). Large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (Squamata: Serpentes). PLoS ONE, 14(5), e0216148.

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Mitochondrial DNA Sequencing of Wild, Captive, and Domestic Individuals

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For 70 wild, 66 captive and 33 domestic individuals we sequenced the mitochondrial DNA fragment NADH dehydrogenase subunit 5 (ND5) based on the study by Driscoll et al. [32] (link). Most juveniles of sampled mothers and siblings were excluded and later assigned to the same mtDNA haplotype as all sequenced juveniles had the same haplotype as their dam. We finally obtained 88 sequences of wild, 77 of captive and 33 of domestic individuals (Tab. S4 in File S1). The primers CD-ND56-F1C and CD-ND56-R4 [32] (link) were used for amplification in a Multigene Gradient Thermal Cycler (Labnet). We used the 5PRIME HotMasterMix (5PRIME) for amplification. The PCR product was purified using the High pure PCR product purification kit (Roche) according to the manufacturer's protocol. Sequencing was performed with the DYEnamic ET terminator cycle sequencing kit (GE Healthcare) for sequencing reactions run on a MEGABACE 1000 automated sequencer (GE Healthcare). Base-calling was performed in Sequence Analyzer 4.0 (Amersham Biosciences).

Witzenberger K.A, & Hochkirch A. (2014). The Genetic Integrity of the Ex Situ Population of the European Wildcat (Felis silvestris silvestris) Is Seriously Threatened by Introgression from Domestic Cats (Felis silvestris catus). PLoS ONE, 9(8), e106083.

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Mapping Genomic Regions in L. major

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To identify the genomic region of interest in L. major genome databases, a 1.0 kb EcoRI fragment from cosTUB1 and a 1.0 kb EcoRV fragment from cosTUB2 were subcloned into a pUC-π vector. The subcloned fragments were sequenced using a MegaBACE 1000 automated sequencer (GE Healthcare, UK) with DYEnamic Dye Terminator kit (GE Healthcare, UK), according to the manufacturer’s instructions. Analyses of the nucleotide sequences were performed using Lasergene Software (DNASTAR, Inc.) and Clone Manager 9 Software. Sequence data for the remaining regions were obtained from LmjF GeneDB [25 (link)]. Nucleotide sequences obtained were used to map the genomic region corresponding to the two different loci using LmjF database. In silico analyses were also conducted to estimate the insert sizes of both cosmids after digestion with different restriction enzymes.

Aoki J.I., Coelho A.C., Muxel S.M., Zampieri R.A., Sanchez E.M., Nerland A.H., Floeter-Winter L.M, & Cotrim P.C. (2016). Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major. PLoS Neglected Tropical Diseases, 10(9), e0004972.

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Microsatellite Genotyping of Orthopteran Species

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DNA was extracted from hind femur muscle tissue using the DNeasy Blood & Tissue Kit (Qiagen). All individuals were genotyped at ten polymorphic microsatellite loci. Six microsatellite markers were designed for Ch. parallelus (BF1, BD5, BH5, BD7, BF9, CD6; Molecular Ecology Resources Primer Development Consortium et al. 2009), four were developed for Ch. montanus prior to this study (Additional file 1). For PCR we used the Qiagen Multiplex Mastermix in multiplexed PCR protocols for a combination of two to four loci with the following annealing temperatures (BF1, BH5, CD6, CM37: 54 °C; BD5, CM5: 48 °C; CM33, CM19: 51 °C; BD7, BF9: 58 °C). PCR tubes were filled with 10 μl reaction mixes (5.5 μl MultiplexMasterMix, 2 μl water, 1.4 μl genomic DNA (2–10 ng), 1.1 primer mix (1 μM/primer). The amplification was performed in a Multigene Gradient Thermal Cycler (Labnet) with the following PCR conditions: Initialization: 94 °C/10 min; Denaturation: 94 °C/45 s; Annealing: see primer/45 s; Extension: 72 °C/45 s; Final Extension: 72 °C/30 min; 37 cycles. Each forward primer was labeled with a fluorescent dye (FAM, HEX or TAMRA). Fragment lengths of PCR products were determined on a MEGABACE 1000 automated sequencer (GE Healthcare) and scored with Fragment Profiler 1.2 (Amersham Biosciences).

Rohde K., Hau Y., Weyer J, & Hochkirch A. (2015). Wide prevalence of hybridization in two sympatric grasshopper species may be shaped by their relative abundances. BMC Evolutionary Biology, 15, 191.

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