The DF/HCC DNA Resource Core provides DF/HCC members, Harvard affiliates, and outside researchers with quality and inexpensive DNA sequencing services and sequenced verified plasmid constructs, paired with comprehensive quality control and excellent customer support. The Core meets the growing need for small- and large-scale DNA sequencing projects, including preparation of DNA from glycerol stocks in advance of sequencing, a plasmid repository and distribution service, fully automated low-temperature plasmid storage, and plasma annotation and informatics support to optimize utilization of the core.
Users of the Core employ sequencing in many types of experiments including to confirm construction of new plasmid vectors, to perform serial analysis of gene expression (SAGE), to confirm the results of site-directed mutagenesis, to determine or confirm the identity of specific cDNA and genomic library clones, and to identify single nucleotide mutations associated with heritable human disease. The Core fills a niche as a moderate-throughput sequencing center that is equally able to manage relatively large and small sample numbers, rather than being restricted to only genome-scale projects. The range of orders the Core has processed for an individual user is from one to thousands of samples. Additionally, the Core has processed samples that were submitted as glycerol stocks, as bacterial pellets, and as purified DNA, with the flexibility of using either standard or user-supplied sequencing primers.
Diverse and large-scale plasmids for research use are available from our PlasmID repository, including ORF collections for bacteria, model organisms, and human. These reagents are supplemented by access to CRISPR-related plasmids though participation in the Millipore Sigma CRISPR core partnership program. Please feel free to contact us with any questions about sequencing or plasmid services.
- DNA Sequencing and Sample Preps
- Plasmid Distribution
- Plasmid Storage
- Additional reagents and consultation through participation in the Millipore Sigma CRISPR core program
Project Title: Development of New Technology for the Study of Membrane Proteins, Protein Complexes, and Viruses
PIs: G. WagnerHMS(Cancer Cell Biology Program)
Detailed analyses of membrane proteins, large protein complexes, and viruses can contribute to the ability to understand the roles of proteins and viruses in cancer cell biology. In this study, Wagner and colleagues improved the current state-of-the-art in nanodiscs used for nuclear magnetic resonance (NMR) by engineering covalently circularized nanodiscs. They then applied the technology to the visualization of a membrane protein and of entry of a non-envelope virus into cells. The technical advance they report has application for both NMR and cryo-electron microscopy (cryo-EM) studies. The report includes a detailed protocol and the plasmid vectors described in the study were of immediate interest to a broad group of researchers.
Contribution of the Core: The Core added plasmid vectors reported in this study to the PlasmID repository, making them broadly available to the research community and alleviating the burden of plasmid distribution from the Wagner lab.
Publication: Nasr ML, Baptista D, Strauss M, Sun ZJ, Grigoriu S, Huser S, Plückthun A, Hagn F, Walz T, Hogle JM, Wagner G. Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods. 2017 Jan;14(1):49-52. PMCID: PMC5199620.
Project Title: Development of a Large-Scale Community Resource for in vivo Study of Cancer Cell Biology and Other Topics
PI: N. PerrimonHMS(Cancer Cell Biology, Cancer Genetics Programs)
Studies in human cell lines have tremendous value but can be meaningfully supplemented by in vivo studies that address questions related to cancer cell biology, metastasis, cachexia, and other topics. Research using fruit fly Drosophila melanogaster has contributed a wealth of understanding relevant to cancer biology and treatments, including discovery of core components of many signal transduction pathways. In this work, Perrimon and colleagues developed a large-scale resource of fly stocks useful for in vivo RNAi studies, including studies in which genes are knocked down in specific stages and/or tissues. The resource is publicly available and opens the doors wide to new studies of cancer biology, other human diseases, and other topics in this exemplary in vivo genetic model system. The fly stocks reported by Perrimon and colleagues have been distributed to more than 2,500 laboratories.
Contribution of the Core: The core provided Sanger DNA sequencing services that were essential for quality analysis of the plasmids and transgenic fly stocks generated in this large-scale resource production effort. The core also distributes plasmid vectors published in this study through the PlasmID repository.
Publication: Perkins LA, Holderbaum L, Tao R, Hu Y, Sopko R, McCall K, Yang-Zhou D, Flockhart I, Binari R, Shim HS, Miller A, Housden A, Foos M, Randkelv S, Kelley C, Namgyal P, Villalta C, Liu LP, Jiang X, Huan-Huan Q, Wang X, Fujiyama A, Toyoda A, Ayers K, Blum A, Czech B, Neumuller R, Yan D, Cavallaro A, Hibbard K, Hall D, Cooley L, Hannon GJ, Lehmann R, Parks A, Mohr SE, Ueda R, Kondo S, Ni JQ, Perrimon NHMS. The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. Genetics. 2015 Nov;201(3):843-52. PMCID: PMC4649654.
If research supported by this core facility results in publication, please acknowledge this support by including the following in your publication(s):
"We thank Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the DNA Resource Core Facility, which provided __________ service. Dana-Farber/Harvard Cancer Center is supported in part by an NCI Cancer Center Support Grant # NIH 5 P30 CA06516."