Maxwell 16

Manufactured by Promega

Sourced in United States, Germany, Italy

The Maxwell 16 is a compact automated nucleic acid extraction system designed for use in the laboratory. It provides a streamlined process for the purification of DNA, RNA, or other biomolecules from a variety of sample types. The Maxwell 16 utilizes pre-filled reagent cartridges and automated protocols to deliver consistent and reliable results.

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Lab products found in correlation

79 protocols using maxwell 16

Total RNA Extraction from K1 Cells

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Total RNA was extracted from K1 cultured cells with the automated system Maxwell 16 (Promega, Madison, WI, USA) after 48 h from transfection following the Maxwell 16 LEV simplyRNA Cells Kit manufacture’s protocol. The quantity and quality of extracted RNA was estimated with Qubit 2.0 Fluorometer (Life Technologies, Foster City CA, USA) by using 2 μl of undiluted RNA solution.

Franceschi S., Lessi F., Panebianco F., Tantillo E., La Ferla M., Menicagli M., Aretini P., Apollo A., Naccarato A.G., Marchetti I, & Mazzanti C.M. (2017). Loss of c-KIT expression in thyroid cancer cells. PLoS ONE, 12(3), e0173913.

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Quantification of miRNA in DCIS Samples

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Samples with pure DCIS (Cohort 2), with adjacent DCIS and IDC (Cohort 3), and with pure DCIS samples from patients with follow-up (Cohort 4), as well the IDC control samples, were laser micro-dissected (LMD) by using the PALM MicroBeam laser microdissector (Carl Zeiss) on 2 μm thick sections. For each sample, 200.000–600.000 μm² were selected and micro-dissected samples were loaded on Maxwell 16 (Promega) to extract RNA, following the Maxwell 16 LEV RNA FFPE protocol. RNA was reverse-transcribed for miR-126 and miR-218 using the TaqMan MicroRNA RT kit (Applied Biosystems). cDNAs in Cohort 2 and 3 were pre-amplified using TaqMan PreAmp protocol (Applied Biosystems) and subsequently droplet digital PCR performed using the QX100™ System (Bio-Rad) according to the manufacturer's instructions. QuantaSoft (Bio-Rad) was used to convert the data into concentrations using Poisson distribution. The miRNAs in DCIS samples from Cohort 4 were quantified by Real-Time qPCR using TaqMan MicroRNA assays (Life Technologies) and a Bio-Rad CFX96™ system. Statistical analysis was performed using the non-parametric median test or Mann-Whitney U test for independent samples (SPSS version 21). Two-sided tests were always used and P values ≤ .05 were considered statistically significant.

Volinia S., Bertagnolo V., Grassilli S., Brugnoli F., Manfrini M., Galasso M., Scatena C., Mazzanti C.M., Lessi F., Naccarato G., Caligo A., Bianchini E., Piubello Q., Orvieto E., Rugge M., Natali C., Reale D., Vecchione A., Warner S., Croce C.M, & Capitani S. (2018). Levels of miR-126 and miR-218 are elevated in ductal carcinoma in situ (DCIS) and inhibit malignant potential of DCIS derived cells. Oncotarget, 9(34), 23543-23553.

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Genomic DNA Sample Preparation for ddPCR

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Cell line genomic DNA was obtained from the Coriell Institute (Camden NJ, NA18507 genomic DNA), ATCC (Manassas VA, LS411 cell line), and Sigma-Aldrich (St. Louis MO, GP2D). Circulating tumor DNA standards were purchased from Horizon Discovery (Cambridge UK). LS411 was purified on a Maxwell 16 instrument (Promega, Sunnyvale CA). BRAF V600E assays consisted of admixtures of NA18507 and LS411. KRAS G12D assays consisted of admixtures of NA18507 and GP2D, except for the KRAS G12D assays against circulating tumor DNA. Before input into each ddPCR assay, cell line genomic DNA was pre-digested with EcoRI (New England Biolabs, Ipswich MA) for 8h at 37C before heat inactivation at 65C. FFPE DNA containing known allelic fractions of BRAF V600E (Horizon Discovery, Cambridge UK) was purified with the Maxwell 16 instrument (Promega, Sunnyvale CA) and admixed together. All genomic DNA was quantified by dye-based fluorescence intensity on the Qubit instrument (Thermo Fisher Scientific, Sunnyvale CA) before performing ddPCR assays.

Lau B.T., Wood-Bouwens C, & Ji H.P. (2017). Robust multiplexed clustering and de-noising of digital PCR assays by data gridding. Analytical chemistry, 89(22), 11913-11917.

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Quantitative UGT2B17 Copy Number Variation

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DNA was isolated from peripheral blood using a semi-automatic nucleic acid isolation method, either on a QuickGene-810 apparatus (Fujifilm, Life Science Products, Tokyo, Japan) or Maxwell-16 (Promega Biotech AB), and quantified on a NanoDrop ND-1000 spectrophotometer (Saveen Werner AB, Malmö, Sweden). The analysis of UGT2B17 copy number variation (CNV) was performed by quantitative PCR on the Mx3000P platform (Stratagene, Cedar Creek, TX, USA), using primers specific for UGT2B17 and for the internal normalization gene GAPDH, as previously described in details (4 (link)).

Mouritsen A., Busch A.S., Aksglaede L., Rajpert-De Meyts E, & Juul A. (2018). Deletion in the uridine diphosphate glucuronyltransferase 2B17 gene is associated with delayed pubarche in healthy boys. Endocrine Connections, 7(3), 460-465.

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Targeted DNA Sequencing of FFPE Samples

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For molecular analysis, 3–10 5–10µm micrometer formalin-fixed paraffin-embedded (FFPE) sections were prepared and tumor tissue was micro-dissected when the tumor content was below 10%. DNA was extracted semi-automated (Maxwell® 16, Promega), and 400ng of input DNA was sonographically sheared (Covaris®) into approximately 200-bp double-stranded fragments. Hereafter, adapters were ligated, and genomic regions of interest were enriched using complementary bait sequences. During this hybrid capture, the selected baits ensure optimal coverage of all relevant genomic regions, including 340 genes in a 1.14 Mb complete genomic territory size. Following the enrichment, the targeted fragments were clonally amplified and sequenced with next-generation sequencing (NextSeq 500/550, Illumina). Point mutations, small insertions and deletions, copy number alterations, and rearrangement/gene fusions were identified with NEO New Oncology’s proprietary computational biology analysis pipeline and analyzed using the NEO diagnosis software.

Schatz S., Falk M., Jóri B., Ramdani H.O., Schmidt S., Willing E.M., Menon R., Groen H.J., Diehl L., Kröger M., Wesseler C., Griesinger F., Hoffknecht P., Tiemann M, & Heukamp L.C. (2020). Integration of Tumor Mutation Burden and PD-L1 Testing in Routine Laboratory Diagnostics in Non-Small Cell Lung Cancer. Cancers, 12(6), 1685.

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Microarray Analysis of Ruxolitinib Response

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For microarray experiments, cells were treated for 4 or 24 h in the presence of DMSO or ruxolitinib (1 µM) and harvested on ice for RNA purification. Total RNA was purified using a Maxwell-16 (Promega) instrument. The experiment was repeated for 3 independent biological replicates prior to microarray hybridization to Affymetrix Human Gene 1.0 ST arrays. Data were normalized in R using RMA and filtered based on variance across all samples and collapsed to the gene level using the genefilter package prior to analysis by one-way ANOVA (for each cell line). P-values were corrected for false discovery rate (FDR) using the Benjamini and Hochberg method⁴⁷. Significant genes (FDR<5%) were contrasted among the treatment groups for each cell line using the Tukey’s post-hoc test. Microarray data have been deposited at the Gene Expression Omnibus (GEO), with accession GSE70508.

Balko J.M., Schwarz L.J., Cook R.S., Estrada M.V., Giltnane J.M., Sanders M.E., Sánchez V., Dean P.T., Wang K., Combs S.E., Hicks D., Pinto J.A., Landis M.D., Chang J.C., Doimi F.D., Gómez H., Rimm D.L., Yelensky R., Miller V.A., Stephens P.J, & Arteaga C.L. (2016). Triple negative breast cancers with amplification of JAK2 at the 9p24 loci demonstrate JAK2-specific dependence. Science translational medicine, 8(334), 334ra53.

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Optimized DNA Extraction from Fecal Samples

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DNA extraction was accomplished by means of the Maxwell LEV Blood DNA Kit (Promega, Madison, CA, USA) with specific modifications to the standard protocol as follows: 100 mg of faeces were tranferred to 1.5 mL clean tube and 400 μL Lysis Buffer were added to the samples. The tubes were mixed by vortexing 60 sec and then incubated for 5 min at 95°C. After that, samples were centrifuged at 13,000 rpm for 5 min. We collected 300 μL of supernatant and transfered to new tubes with 30 μL Proteinase K solution followed by a second incubation step at 56°C for 20 min. The total volume was loaded into the cartridges provided by the kit for the final automated step in the Maxwell 16 instrument (Promega, USA). DNA was quantified by Qubit Fluorometer 2.0 (Thermofisher Scientific, Waltham, MA, USA) using the Qubit dsDNA HS (high sensitivity) Assay Kit (Thermofisher Scientific, USA).

Ianni A., Bennato F., Di Gianvittorio V., Di Domenico M., Martino C., Colapietro M., Cammà C, & Martino G. (2022). Impact of different shades of light-emitting diode on fecal microbiota and gut health in broiler chickens. Animal Bioscience, 35(12), 1967-1976.

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Whole-Genome Sequencing of P. aeruginosa Isolates

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Nine previously described environmental isolates of P. aeruginosa (Feltman et al. 2001 (link)) were selected for sequencing. These isolates were streaked from −80 °C frozen stocks, inoculated in Luria-Bertani broth, and grown with shaking overnight at 37 °C. Genomic DNA was extracted from the cultures using the Promega Maxwell 16 instrument (Madison, WI) according to the manufacturer’s instructions. Genomic DNA was sequenced on the HiSeq 2000 platform yielding 101-bp paired-end reads. To maximize assembly quality (Wall et al. 2014 (link)), each paired read set was randomly downsampled to obtain estimated 80-fold genome coverage and de novo assembled using Ray v1.7.0 (Boisvert et al. 2010 (link)). Assembled contigs smaller than 200 bp were removed from the analysis. Contig sequences were deposited in GenBank under assembly accession numbers GCA_002239415.1, GCA_002239425.1, GCA_002239445.1, GCA_002239465.1, GCA_002239485.1, GCA_002239505.1, GCA_002239535.1, GCA_002239545.1, and GCA_002239565.1.

Ozer E.A., Nnah E., Didelot X., Whitaker R.J, & Hauser A.R. (2019). The Population Structure of Pseudomonas aeruginosa Is Characterized by Genetic Isolation of exoU+ and exoS+ Lineages. Genome Biology and Evolution, 11(7), 1780-1796.

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Extraction and Sequencing of Microbial DNA from Buccal Swabs

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DNA was extracted from Epicentre Buccal Swabs and purified using the Maxwell 16 Buccal Swab LEV DNA Purification Kit (Promega AS1295) and the Promega Maxwell 16 (AS1150), set on the standard bacterial protocol extraction setting for the machine. Purified DNA samples were then loaded into the wells of 96 well plates at 30 µl and a minimum of 10 ng/µl per sample. We left four blank wells for sequencing controls and the plates were sealed and stored at −20 °C. Samples were shipped on dry ice to the Knight Lab at the University of California San Diego for amplification and sequenced using an Illumina MiSeq, targeting the V4 region of the 16S rRNA gene using modified 515F–806 R primers as recommended by the Earth Microbiome Project^{64 (link)}. The V4 region was also chosen based on its nearly universal bacterial and archaeal annotation and availability for alignment in reference databases such as Greengenes^{65 (link)}.

Burcham Z.M., Garneau N.L., Comstock S.S., Tucker R.M., Knight R, & Metcalf J.L. (2020). Patterns of Oral Microbiota Diversity in Adults and Children: A Crowdsourced Population Study. Scientific Reports, 10, 2133.

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Extraction and Purification of Genomic DNA

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Genomic DNA (gDNA) was extracted from 100 mg (soy) or 200 mg (maize) ground dry material with the Maxwell 16 instrument (Promega, Mannheim, Germany) using a modified protocol [17] . Some batches of isolated gDNA were further purified with DNA Extractor Cleaning Columns Kit (Eurofins-GeneScan). Genomic DNA was not enzymatically digested prior to ddPCR if not otherwise indicated, plasmids were purchased linearized. Extracted DNA was either frozen at −20 °C for long-term storage, or kept in the fridge at around 5 °C for usage within days.

Gerdes L., Iwobi A., Busch U, & Pecoraro S. (2016). Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms. Biomolecular Detection and Quantification, 7, 9-20.

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