... similar brightness - even though in the most popular pair, CFP has five-fold less brightness than YFP...Master. Note: BRET= Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer...that can affect FRET measurements include: the brightness of a fluorophore pair, donor:acceptor stoichiometry...

... are essential for processes such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction...reactive and forms a link with nearby nucleic acids (McInnes et al., 1990). Photobiotinylation is sometimes ...18988027. PubMed Central PMCID: PMC3223083. 6. McInnes, J. L., Forster, A. C., Skingle, D. C., & Symons...Biotinylation ThermoFisher Scientific: Biotinylation Guidelines  ...

...Isolation and Concentration (MagIC)-cryo-EM that combines enrichment and imaging of proteins captured on...with a GFP nanobody (Figure 1). This enrichment streamlines the purification and minimizes sample loss while...loss. Ci Ji Lim’s lab found that adding Late Embryogenesis Abundant (LEA) proteins, small proteins that... to partially correct mouse models of genetic blindness (An et al., 2024) — the first demonstrated use...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...

...Repair is Used to Edit Genes Read How Non-Homologous End Joining is Used to Delete Genes Additional Resources...exons), but much of the sequence appears to be nonessential. Within the mutational hotspot for dystrophin... cells expressed functional dystrophin. Editing genes in vivo is of course much more difficult than in...these different mutations. For each therapy, effectiveness and off-target risk must be evaluated separately...

...plasmid yield and/or DNA quality. Table 1 below outlines a few of the more common genetic changes found...encodes proteins needed for bacterial conjugation. Genes listed on F´ are wild-type unless indicated otherwise...lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced, expression...transformation efficiency deoR constitutive expression of genes for deoxyribose synthesis Allows uptake of large...)]  Note: Inactivating mutations in specific genes are signified with a minus sign (-) as is typically...

...Finally, as Pamela Hines of AAAS points out on the panel, irreproducibility undermines public trust in science...Last Wednesday we worked with the Harvard GSAS Science Policy Group to organize a Minisymposium on Reproducibility...) - Panel Alex Tucker (Ginkgo Bioworks) Pamela Hines (Senior Editor at Science) Edward J. Hall (Professor...journalists and scientific experts discussed by Pamela Hines Minisymposium on Reproducibility Booklet - Including...

...edits and to perform functional screens to identify genes involved in a phenotype of interest. This blog post...protein-coding capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both...Veres et al., 2014). When working with single-cell clones, the authors note that “clonal heterogeneity may...obstacle to the generation of truly isogenic cell lines than nuclease-mediated off-target effects.” Further... CRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing. Cell Stem ...

...involves making a large sgRNA library to thousands of genes and targeting and sequencing millions of cells. ...cell line, with the goal of isolating single cell clones to use for multiple downstream experiments. You...certain Cas enzymes are less compatible with the bulkiness that accompanies mispairing of bases. To reduce... characterization of, isolating 2 or 3 distinct clones to perform key experiments on is a good way to ... distinct off-target sites, eliminating its effectiveness as a control. Since off-targets typically confound...

...al., 2009), and at high copy numbers (Williams, Carnes, and Hodgson, 2009). More research is ongoing to...1566524020666200427110452. PMID: 32338220. Crozat E, Fournes F, Cornet F, Hallet B, Rousseau P. Resolution of...Levene SD. Analysis of DNA topoisomers, knots, and catenanes by agarose gel electrophoresis. Methods Mol Biol...0092-8674(84)90060-6. PMID: 6323019. Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design... JA, Luke J, Langtry S, Anderson S, Hodgson CP, Carnes AE. Generic plasmid DNA production platform incorporating...

...many of which are used to deliver Cas9 and other genes to mammalian cells. For this reason, the top requested...means researchers who use VSV-G can deliver their genes of interest to a variety of cells and study their...Skimming the list, one gets a sense of the broad usefulness of pMD2.G and VSV-G. This plasmid has been used... of the secretome, ubiquitin ligase, cellular senescence, autophagy, CRISPR, and so much more (find the...

...societal implications even before the discovery of genes themselves.  How has scientific progress in genetic...and vice versa? We draw a parallel between the timelines of the scientific advancements and the noteworthy...to stars in space ships and the rise of killer machines that exterminate entire humanity were the two ...that results in gene knockouts.   Star Trek: Into Darkness (2013) which is the remake of the space-melodrama...Hugo De Vries: from the theory of intracellular pangenesis to the rediscovery of Mendel. Comptes Rendus ...

...Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh promoter. Cbh is a modified...Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component of most CRISPR...gene that made it less effective in certain cell lines. This point mutation has been corrected in V2.0....gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain GFP-tagged...

...overview of the instrumentation A flow cytometer combines three systems to analyze single cells from a mixture...degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression, measured...targets, measured by fixing and permeabilizing membranes For intracellular protein targets, users must ...propidium iodide, can only enter cells with disrupted membranes as is the case with dead or dying cells. DNA binding...the cells, amine reactive dyes interact with free amines in the cytoplasm causing dead cells to fluoresce...

...visualize the protein via fluorescence or chemiluminescence, precipitate the protein out of solution, ...region   The constant region of the heavy chain determines the antibody’s isotype - that is, one of five...which come from naturally-occurring immunoglobulin genes, recombinant antibodies are plasmid-based. Their...antibody allows the protein to be detected by chemiluminescence or fluorescence. Western blots are frequently...Steven GE, Lorna J. (2004) ANTIGENS, IMMUNOGENS, VACCINES, AND IMMUNIZATION. In: Immunology Guidebook. Elsevier...

...screens using CRISPR technology, gRNAs to knockout genes – are essential screening components, the model ...Sometimes, you might have a few positive control genes on which to test the validity of your biological... a new assay or both, and you have no idea what genes are involved. Or you don’t know how well your assay...definition, the kinome is a well-annotated collection of genes that can provide a useful starting point for developing...

...Directed Mutagenesis by PCR Google Forums Round Up: First Impressions of NgAgo CRISPR Between the Genes: How...optimize and troubleshoot the system. Site Directed Mutagenesis by PCR & Single Base Editing with CRISPR For ...technology may make some uses of Site Directed Mutagenesis by PCR obsolete! Honorable mentions Of course...

...miss out on beneficial opportunities or to find oneself unsure of the next steps. Harvard PI Angela DePace...lab culture. The aim of these meetings is to give honest, constructive feedback, set tangible research and...personal development through thoughtful planning and honest feedback DePace’s yearly planning meetings revolve...person organize his or her thoughts to facilitate honest conversation. Goals and Planning Worksheet: ...

... more often than other disciplines. The majority of tweets for most disciplines contain scientific domains...#8. Figure 1: Highly Represented Scientific Disciplines on Twitter. Twitter users were identified as ...Follower networks of subgroups of scientific disciplines: social (left), life (center), and physical (...

...based on a protein isolated from Streptococcus pyogenes that locks itself together, to produce irreversible...another important step forward by dissecting that S. pyogenes protein into three parts. Their efforts yielded... from the “S” in Streptococcus and the “py” in pyogenes), capable of locking two peptide tags together... chain.  Addgene: What about the Streptococcus pyogenes protein domain made it ideal for this purpose?...

...into the following categories: Over 300 empty backbones with 100+ distinct fluorophores to choose from...constructs for biosensing. Over 300 plasmid backbones spanning a rainbow of fluorescent proteins “It...It’s a great collection with over 300 backbones alone,” said Addgene’s Lianna Swanson, who has been working...collection with such variety.” In most cases, backbones are available to attach the gene of interest to...