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  1. CRISPR 101: Epigenetics and Editing the Epigenome

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    ...epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting...DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic...Preps for Epigenetic Modification Why use epigenetic modifiers? Epigenetic modification is certainly not ...Nature Communications 8: . https://doi.org/10.1038/ncomms16026 Find plasmids from this publication at Addgene..., 2017 and updated Jun 24, 2020. Epigenetic modifications are an additional layer of control over gene...efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression...mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR...
  2. Plasmids 101: Protein tags

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    ...or DYKDDDK 1 Affinity and Purification Good for antibody-based purification; has inherent enterokinase...site GST Large Protein 26 Purification and Stability Good for purification with glutathione; protects...used for purification as elution requires low pH poly His HHHHHH  0.8 Affinity and Purification Very small...GKPIPNPLLGLDST   1.4 Affinity and Purification Good for antibody-based purification Combo and cleavage tags...increasingly common. Tandem affinity purification (TAP) once referred specifically to a combo tag comprised of ...multitude of uses including (but not limited to) purification, detection, solubilization, localization, or...overexpress and purify proteins can face any number of technical challenges depending on their protein of interest...
  3. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

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    ...differing in pigmentation." Biochemical and biophysical research communications 78.1 (1977): 237-243. PubMed...one that can be achieved using pharmacological or electrophysiological methods (Deisseroth, 2011; Mei ...recombination. Purification and properties of the Cre recombinase protein." Journal of Biological Chemistry...Edward S., et al. "Millisecond-timescale, genetically targeted optical control of neural activity." Nature ..., et al. "Next-generation optical technologies for illuminating genetically targeted brain circuits." ...and Jinfang Zhu. "Immunologic applications of conditional gene modification technology in the mouse." Current... R. Thompson. "Overview on research and clinical applications of optogenetics." Current protocols in pharmacology...
  4. Magnetic Control of Proteins: More than a Dream

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    ...Spatially Confined Persistent Diradical Emitter. Journal of the American Chemical Society, 145(25), 13615–13622... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible....of developing new microscopy tools for biological applications across a wide range of topics. He embraces...., Timmel, C. R., & Hore, P. J. (2016). Chemical amplification of magnetic field effects relevant to avian...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...tool with magnets would enable new research and applications that don’t yet exist, but it wasn’t clear that...
  5. Live and Let Dye: Self-Labeling Protein Tags

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    ... construct for every application. Plus, the photophysical properties of chemical dyes provide many advantages...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic... W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research, 44...If you take a look in a typical lab freezer, you’ll probably find lots of different plasmids containing...new green fluorescent protein, some affinity purification tags, another version with that same red fluorescent...substrate. If the substrate was linked to, say, a chemical dye or other functional ligand, your protein of...advantages over fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies...
  6. Plasmids 101: Broad Host Range Plasmids

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    ...of Various Motifs is Critical for Initiation of Replication. Journal of Biological Chemistry, 273(14), ...to utilize diverse host replication machinery. For instance, the ori typically contains iterons that serve...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts...the ori and allow for replication. However, if plasmids have their own replication machinery such as Rep...different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow... while others have a broad host range and can replicate and stably maintain the genes they carry among.... These features are: The plasmid origin of replication (ori) contains structural elements that are versatile...
  7. Typing CRISPR Systems

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    ... your own research! Classification Structure Principles of CRISPR classification While CRISPR systems... CRISPR-Cas systems. Philosophical Transactions of the Royal Society B Biological Sciences, 374(1772),...Shakespeare for the first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied...properties. This led to a need to develop a classification system for the array of CRISPR technologies...technologies. Learning the general principles of this classification system will help you gain a deeper appreciation...multi-pronged analysis to sort effectively. The current classification system relies on a combination of sequence ...a similar function. After analysis, CRISPR classifications were placed into a fairly simple hierarchy:...
  8. Better Dyeing Through Chemistry & Small Molecule Fluorophores

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    ...interaction between a bisarsenical dye ligand (e.g., FlAsH, ReAsH) and a short genetically encoded tetracysteine...systems consist of a genetically encoded enzyme variant tag that reacts specifically and irreversibly with... as intact tissue. Some chemical labeling strategies such as the bisarsenical dye (Figure 1a) and FAP ...Ronald T. Raines. "Bright ideas for chemical biology."ACS chemical biology 3.3 (2008): 142-155. PubMed... at the Janelia Research Campus, Howard Hughes Medical Institute. Chemistry is dead, long live chemistry...fluorescent protein (GFP) sparked a renaissance in biological imaging. Suddenly, cell biologists were no longer...synthetic fluorophores. Add a dash of DNA with an electrical jolt and cells become perfectly capable of synthesizing...
  9. Delivery Methods for Generating iPSCs

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    ...Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect...lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox lentiviral vectors:... to reprogram. Basically, there’s no universal method that can handle all applications of iPSCs, but there...generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem cell reprogramming... to verify there’s no genomic integration Replicating EBNA1 episome Non-integrating Medium Medium ...creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing...unavoidable heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene...
  10. Isolating Genomic Regions of Interest with the CRISPR System

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    ... (enChIP) using CRISPR." Biochemical and biophysical research communications 439.1 (2013): 132-136. PubMed...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...26848818. Fujita, Toshitsugu, et al. "Identification of physical interactions between genomic regions ...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...
  11. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

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    ...transfected (see Technical Tips). Minigenome replication and transcription In order to replicate the minigenome...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...that are crucial cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome...5’ ends of the genome which are required for replication and transcription (Figure 1). All viral genes...cis-acting signals necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase... in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...
  12. When Fidelity Matters: A frank discussion about ligase fidelity

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    ...resistance-mediating polymorphisms in African samples of Plasmodium falciparum." Journal of clinical microbiology 51.8 (...furiosus." Acta Crystallographica Section F: Structural Biology and Crystallization Communications 61.12 (2005... In vitro, ligases (notably T4 DNA ligase) are critical reagents for many molecular biology protocols,...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...rounds of melting and annealing allows linear amplification of the probe ligation product. With the right...
  13. Healthcare Consulting: A Door to the Business of Life Sciences

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    ...D) teams at pharma companies carry out preclinical and clinical research to discover and test the best...years. Complicating these challenges is the fact that they occur in the context of significant clinical...training in reviewing literature, and critical analyses skills were applicable to my cases. I can directly use...molecular behaviors of chemicals and proteins to learn how a particular drug or medical device works. This... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance...efficient ways of delivering healthcare. Clients typically engage consultants as short-term experts to work...acquisitions of or collaborations with other companies. Medical Affairs groups work on building awareness among...
  14. 22 Hot Plasmid Technologies from 2014

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    ... receptors which play a critical role in development and are often implicated in disease progression. ...inability to replicate the spatio-temporal precision with which signaling events occur in a physiological setting...brighter mutant S65T-GFP, and the subsequent biochemical characterization, has enabled scientists to engineer...observations: Rapamycin is a membrane-permeable pharmacological agent that can rapidly induction heterodimerization...flexible cloning process provides improvements to classical cloning methods, particularly for complex transgenesis...enabled the group to perform independent two-color optical excitation of neurons. The paper describes five...setting. Genetic techniques typically rely on overexpression or knock-down, where signaling dynamics are...
  15. Viral Vectors 101: Systemic Capsids

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    ...rather than being geographically constrained to the brain. In cases where it’s critical to minimize or eliminate...recommended for all systemic capsid applications. For example, AAV-PHP.eB is typically administered at a total dose...your specific application and animal model.  Other model organisms Tropism and efficacy can vary depending... 2023 * indicates viral preps available. Mechanisms of crossing the BBB are indicated in parentheses...crossing into the brain. While helpful from a biological standpoint, it makes delivering AAVs to the brain...throughout the organism to target large or diffuse biological structures. Of primary interest here are systemic...suitability of a systemic capsid for an individual application is dependent on a number of factors, including...
  16. 27 Hot Plasmids from 2016

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    ...FAIRE-seq, and CHIP-seq), genome-wide identification and quantification of enhancer activity have remained...Identifying new ligands for orphan GPCRs is of significant medical interest, however efforts to do so have been...GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones for lentivirus...mutant library generation in several biotechnologically and clinically relevant bacterial species. pORTMAGE...backgrounds can lead to disparate physiological effects and complicate any metabolic study. The Ralser ...brief articles highlight the main features and applications of a partiular plasmid or set of plasmids. We...Wu Lab library can be found in the associated publication. Ma, et al. Cell Rep. 2015. PubMed PMID: 26190106...
  17. GCE4All: Making Genetic Code Expansion Accessible

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    ...protein site-specifically to alter its function, mimic a biologically relevant modification, or add in ...function relationships or, in more practical applications, allow a protein to be more effectively used in ...presents a significant barrier for those wishing to use it as a tool. In short, while the technical skills...proteins, you’re probably quite interested in the canonical 20 amino acids. But in your quest to learn more...code expansion (GCE): techniques in which non-canonical amino acids (ncAAs, also sometimes called unnatural...in fluorescent or chemically reactive side chains, all of which can help determine structure and function...was developed into an E. coli system, with the noncanonical amino acid in the media and the tRNAs and synthetase...
  18. Fluorescent Proteins 101: When GFP lets you down

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    .... "A genetically encoded fluorescent probe in mammalian cells." Journal of the American Chemical Society...). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several factors contribute...probe, it is the first choice when selecting a genetically encoded fluorescent tag. There are, however, ... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...of these strategies works best for a specific biological system under anaerobic conditions needs to be...adding GFP may alter diffusion rates. Smaller genetically encoded probes are based on cofactor binding ...tags are peptides that are engineered to bind specifically and with high affinity to fluorophores. Examples...
  19. Summer SciComm: Preprints

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    ...or feedback. One of the most significant shifts in scientific communication is the rising use of preprints...arXiv.org), but have been only adopted by biological and biomedical scientists (see: bioRxiv.org or medRxiv.org...the most important part of your research is communicating the results of the work. Yes, those control ...any impact! There are lots of ways scientists communicate with each other: from formal mechanisms like ...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, social...publishing in a journal.  Accelerating scientific communication Preprints have been common in fields like math...increasingly growing time from submission to publication. It takes many months, often more than a year...
  20. Troubleshooting and Optimizing a Western Blot

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    ...people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant...background, your issue is likely technical. White circles, for example, could indicate that you had air trapped...Sensitivity in Western Blotting. Journal of Pharmacological and Toxicological Methods, 64, 233–237. https://doi....to get right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself... be done at 4 °C or on ice, whichever is more practical. If your lysing method is not resulting in your...nuclear- or DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins ...you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you...
Showing: 201 - 220 of 1082 results