... regions of homology at their ends, which are typically created by PCR. Then, the fragments are incubated...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...competent cells. The success rate of this reaction is typically fairly high, so there is usually no need to screen...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...the bacterial chromosome (up to 100 kb) were specifically cut out via CRISPR and then assembled into a...

...improve our shipment processes. After reading a publication describing how short term storage on dry ice ...aqueous samples might be affected by dry ice, and specifically how dry ice can affect virus from Addgene. ...medium, which is buffered and contains the pH indicator phenol red. We froze samples in a -80°C freezer...closed dry ice storage container for 48 hours (to replicate how our viruses are shipped), where they would...exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or rather, not ... the samples were at the control pH of 7.4 as indicated by color (data not shown). What we did next: Venting... at -80°C. We then thawed the tubes after the indicated incubation periods, and measured the pH. We found...

...Talley Lambert, a Research Associate at Harvard Medical School. The need for a community fluorescent protein...one of the most indispensable tools in modern biological research.  Any microscopist will tell you that...non-trivial, however, as FPs are tremendously complicated entities with a large range of characteristics...maturation, oligomerization), many of which are dramatically affected by environmental conditions (such as...decision remains spread across literature in publications that introduce these tools. Years ago, Kurt...incorporate FPbase data in their own projects and applications, a REST API allows programmatic retrieval of...Talley Lambert is a Research Associate at Harvard Medical School. He manages the Cell Biology Microscopy ...

...transcription, or DNA repair, or replication, or any number of CRISPR applications and thought “OK, but how does...pages containing lists of plasmids from the indicated publications. You can request all of these plasmids ...to think about DNA in this context, but also specifically study it. They might be researching enhancer...--directly or indirectly--many other areas of biological research. If you’re looking for resources that...you study how chromatin effects your favorite biological phenomenon, there’s a reasonable good chance ...below pull up lists of plasmids containing the indicated gene, and you can subscribe to these pages to ...RANbodies) as versatile, small, sensitive immunohistochemical reagents. Yamagata M, Sanes JR Proc Natl ...

...proof-of-concept for pre-clinical and clinical applications of dCasRx. The identification of Type VI Cas13d ...RspCas13d. RspWYL1 also increased EsCas13d activity, indicating that WYL domain-containing proteins may be broader...even when guides were tiled across a target RNA, indicating that this enzyme does not cleave at a predictable...efficiency; where shRNA treatment produced 500-900 significant off-targets, CasRx displayed zero. Unlike Cas9...target essentially any RNA in a cell. Since catalytically dead dCasRx maintains its RNA-binding properties...enhancers. After AAV-mediated delivery to iPS-derived cortical neurons, dCasRx-mediated exon skipping improved...

...comparable in efficacy to dCas12a systems in both gene activation and base editing applications.  Figure...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...members or students new to protein expression and purification. Figure 3: The Penn State Ladder run ...challenges for in vivo delivery and cell engineering applications. CasMINI, a new miniature CRISPR-Cas system ...: Overview of CasMINI for genome engineering applications. Image from Xiaoshu, et. al. 2021.   Created...

...population (typically cells or small organisms) and then assaying for a functional or phenotypical outcome... daunting task of finding new factors in your biological system of interest, the so-called “fishing expedition...that both attempt to identify new players in a biological process. Players can be proteins, genes, enzymes...obligatory growth advantage – that is, you are not specifically looking at cells that can survive. Instead, ..., the entirety of the small molecule realm is typically not feasible to test in any given screening experiment...selection process. Non-functional traits will be automatically eliminated, unlike in screens. Selections begin...the answer that much faster? In reality, many biological questions are not conducive to selections and...

...their idea is. While communication styles in academia and industry vary drastically, he reassures that ... As a biomedical engineer and scientist, I like to envision that my research will have a direct impact...competitions. After much learning, the focus of the application was changed, but Sample6 still leverages the ...fit for this set of technologies, effectively communicating the advantages embodied, and driving the enterprise...He explained that entrepreneurs must clearly communicate their idea at various levels of complexity and...biggest challenge during this time was learning to communicate effectively to non-scientific audiences. "There...for the company; this will help avoid future complications and potential conflicts that waste money and...

...communities aim to significantly improve the reproducibility and reliability of science publications. Peer reviewing...should I choose? Writing a research paper for publication in a scientific journal requires several considerations...Information Framework (NIF) called the Resource Identification Initiative. During this project, authors are...likely that researchers have enough information to replicate your experiments, making research more reproducible...happens before the manuscript is accepted for publication and is, in most cases, blind (meaning you don...revised before it can be published, this can delay publication and manuscripts may go through several rounds...a novel form of peer review that occurs post-publication and is completely open; the reviewer’s name and...

...Inagaki, S., et al. (2017) Genetically encoded bioluminescent voltage indicator for multi-purpose use in...et al (2). In this post, I’ll cover two great applications of the NLuc technology. The first couples NLuc... requires long acquisition times that may be impractical or infeasible for certain experimental setups... phototoxicity, autofluorescence, and perturb biological phenomena. The technique of coupling FPs with...fluorescence. The paper reports a wide variety of applications of the Enhanced Nano-Lanterns including monitoring...can be correlated to specific concentrations of biological molecules. FRET sensors face challenges of photobleaching...membrane-inserted voltage sensing domain (LOTUS V; Luminescent Optical Tool for Universal Sensing of Voltage). With this...

...expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often be proximal... to keep in mind, but likely will not have a significant impact on your data. You are trying to generate...product with CRISPR to (hopefully) be used in human clinical trials. An off-target rate of 5% is far too high...Cas-OFFinder, CCTop, and many more. While such software typically only considers DNA sequence and doesn’t take into...sequencing. While this may be unnecessary for many applications, it is the only comprehensive method available...selectively sequenced to determine if there are significant editing events, serving as a proxy for total ...For a candidate site-specific approach, mRNA quantification or western blot of putative off-targets would...

...Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neuroscience...Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific critical dynamics triggering perception. Science... activated tools, can control everything from electrical activity to gene expression, and even CRISPR ...through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been...stimulation. GPCR signaling GPCR signaling is a complicated process involving many downstream signaling mechanisms...break your experiments!  First, closely read the publication, including the supplemental data. You’re most... both of which have extensive collections of publications and information on hardware, optogenetics tools...

...target specific tissues, even when administered systemically, without causing disease? Recombinant adeno-...in-stock viral prep service.  AAV5 AAV5 is the most genetically distinct AAV. Though AAV5 is one of many serotypes...vessel barriers. It is especially popular for applications targeting liver tissue. In addition, it has ...the blood-brain barrier, making it useful for applications targeting the CNS (Issa et al., 2023). Additionally...al., 2019 Liver AAV8 Sands, 2011 Lung pUCmini-iCAP-AAV9.452sub.LUNG1 Goertsen et al., 2022 Pancreas...treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes varies by...on the AAV capsid. Molecular Therapy. Methods & Clinical Development, 26, 331–342. https://doi.org/10.1016...

...variety of research applications, including making transgenic cell lines and genetically-modified cell lines...facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed in the genome...piggyBac™ technology can be used for numerous applications, including transgenesis, gene-trap screens, ...of interest. The piggyBac™ system allows for significantly enhanced random integration efficiencies that... If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...of the piggyBac™  transposase/transposon gene modification system and the scarless, Footprint-Free™ removal...re-integration from the piggyBac™ donor, which is typically found with the wild-type piggyBac™ transposase...

...since other signaling networks can also be non-specifically activated and, like the game of telephone, often...must be transmitted by intermediate messengers. Optical control of kinase activity can provide greater ...greater spatiotemporal resolution than pharmacological or genetic approaches, but only a few such methods exist...Problem:  There is no common method for creating optically controlled kinases. The Solution: In Zhou et al...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...kinases good for? There are a number of potential applications for photoswitchable kinases.  Below are a few...endogenous ERK, and its localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates...

...degraders provide distinct advantages over pharmacological inhibitors including rapidly depleting a protein-of-interest...resistance to inhibitors, and inducing prolonged biological effects. While targeted degradation of endogenous...here. The Methods section of Nabet et al., Nature Chemical Biology additionally provides detailed information...FKBP12F36V binding ligand (ortho-AP1867) via various chemical linkers. Our current lead dTAG molecule is dTAG...FKBP12F36V-tagged LACZ) with dTAG molecules in your biological assay of choice to ensure that there are no toxicities...µM (see Figures 3 and 5 in Nabet et al., Nature Chemical Biology for examples). Monitor neo-substrates ...experiments (Nabet et al. and Weintraub et al.), chemical and genetic rescue experiments (Nabet et al.),...

...pathways and how they lead to neurological phenotypes observed.” Using genetically-encoded biosensors and iPSC-derived...Scientific Advisor. It’s possible that solute channels typically expressed on other cell types can compensate for.... “This proof of concept study will help in identification of new targets that could greatly enhance our...understand so that we could possibly develop pharmaceutical interventions that would inhibit IL-36 release...genes from the X chromosome are expressed at significantly reduced levels in women with TS (Trolle et al...Terry Hébert, a professor and assistant dean of biomedical science education at McGill University.  These...58 individuals with GNB1 disorder since the identification of this disease in 2016. The lab aims to “expand...

... antibodies may be indicated on occasion, usually after other signal amplification techniques have been...IgG2 or IgG4. This can be important for some applications. Assay use While secondary antibodies are useful..., the flexibility of their use varies across applications. Western blots, for instance, almost always ...For the latter three, secondary antibodies are indicated when the protein of interest is found in low amounts...been ruled out.  Ease of access Signal amplification isn’t the only benefit of secondary antibodies. Finding...which means that indirect methods are often significantly longer than direct ones.  Even though the secondary...quantitative analysis of a sample. Instead, relative quantification can be done using either the direct or the ...

...from the serum. Protein A/G purification Protein A or Protein G purification is one common method. These... the desired level of antibodies is reached - typically within 2-4 months. After the animal has the desired...reagent that works for your species. Affinity purification Only up to 5% of the IgG antibodies produced...specific for the immunogen. A different type of purification can be performed to isolate antibodies only ...inexpensive. Their short production time and the bulk purification steps keep the cost low compared to other antibody...are particularly useful for certain research applications. Many secondary antibodies are polyclonal, as...target protein thereby greatly increasing signal amplification.  When to stay away from polyclonal antibodies...

...and can be specifically targeted to a number of different cell types even when systemically administered... of AAVs contain two numerical designations, separated by a slash, that indicate the ITR and serotype,...experiments. Recombinant AAVs (rAAVs) The primary application of AAV is delivery of genetic cargo to a host...with maximum “trunk” space for genetic cargo. Typically, when researchers speak about AAVs, they are actually...transduced gene is episomal and does not divide and replicate in the same way the host DNA does. Because of ... since you don’t have to wait for the host to replicate the DNA before expression. Now that you’ve got... S., et al. “Various AAV Serotypes and Their Applications in Gene Therapy: An Overview.” Cells, 12(5),...