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...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...grow overnight cultures for DNA purification. The simplest purification you can do is a miniprep, but if... maxiprep. In these purifications, you generally lyse the bacteria; add chemicals to precipitate out the...that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of...digests. Of course there’s much more detail and verification required for the process to work well, so let...backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...

... microscopy technique to incorporate optical sectioning. Optical sectioning refers to the ability to extract...Super-resolution microscopy uses a number of optical and chemical ‘tricks’ to turn subsets of fluorescent ...Richardson, Director of the Harvard Center for Biological Imaging and a Lecturer on Molecular and Cellular...are a sports photographer at the Super Bowl, a medical technologist taking an x-ray, or a biologist imaging...excitation volumes to enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence...before reaching the detector. The pinhole is specifically placed to only allow light from the focal plane...of a sample. Therefore, when a large number of optical slices are acquired all fluorophores throughout...

... sport athletes, as well as the ethical and health policy implications arising from sport-related concussion... years of exclusively writing lab reports and empirical essays had also stirred some of my more creative...begun my journey towards a career in science communication, an established field of inquiry with which ...University of Otago’s Department of Science Communication the following year and, having thoroughly enjoyed...straight on to a master's degree in science communication. The first year of the course involved a whirlwind...world of industry. Along this road, science communication graduates are often well-equipped. Many institutions...truly benefit others. Herein, a good science communicator—with an understanding of how to appeal to a ...

...transmitted transsynaptically). This is because the recombinant forms of these viruses typically used in ...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...tracing, it has two practical limitations. First, these high levels of viral replication are toxic. Second... well as to only spread monosynaptically from these cells. These modifications take advantage of the fact... became foundational later on when we studied biological processes, like how our stem cells are housed... thus, are a major part of the answer to many biological questions about the brain. To address this, molecular...enter cells and deliver a variety of cargoes, genetically engineered neurotropic viruses emerged as powerful...

...desired modification. Finally, the positive clones are sequenced to confirm the desired modification and ...polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification fidelity), and no... genome modifications. Occasionally, multimerization of the PCR primers can cause duplication of the primer...the desired modification have been identified (and confirmed to be free of primer duplications), final validation...used to mutate the PAM sequence (an NGG sequence critical for Cas9 cleavage), thereby rendering the resulting...and the absense of additional modifications.   Site-directed mutagenesis of Plasmids. A) Generation..., which flank the targeted region. Upon PCR amplification, the template is removed by DpnI restriction...

...recombination. Yeast Replicating plasmids (YRp):  These vectors contain an Autonomously Replicating Sequence (ARS...These vectors replicate as though they are small independent chromosomes and are thus typically found as a...not necessarily promote replication.  Selection markers for yeast Historically, scientists have utilized...mammalian vectors, will catch you up on the core replication and resistance features of yeast vectors and ...proteins yet they still share many of the same technical advantages that come with working with prokaryotes...includes but is not limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic...mutation. Yeast expression plasmids used in the lab typically contain all the necessary components to allow ...

...systems have been used as biological tools for a wide range of applications in molecular biology or in...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...counterselectable marker to create unmarked genetic modifications in the yeast Pichia pastoris. By combining both...

...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...episomal vectors is to include viral oris to drive amplification, which helps increase cellular uptake, but this...

...a tail, and when gap junction-mediated electrical communication is briefly inhibited in the entire worm...fluorescent reporting and genetically encoded fluorescent voltage indicators (GEVI) are ideal tools to...thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal cells ...It's just that for non-neural cells, ion-based communication happens at a slightly slower scale.  All living...living cells with a membrane possess an electrical state. This state, known as the resting potential (... (Vmem), can be measured as the imbalance of electrical charge between the inside and the outside of the...bioelectricity is revealing the important role of bioelectrical signaling mediated by ion-channels and pumps...

...Each round of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification... Purification is required if rAdV is to be used in vivo. The standard method for purification of rAdV ...or 10 days) Physical titer (particle count, rAdV genomes/mL) can be measured by: Optical density: particle...vectors (AdV) are attractive vectors for research applications and gene therapy: they can be produced at high...AdVs is what makes them ideal candidates for applications in oncolysis and vaccination!) In the 65 years...years since its discovery, researchers have done significant work to improve the Adv genome’s utility in research...Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new...

...are currently approved or have made significant progress in clinical trials.     Figure 1: Types...editor-based clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for...choice. In sum, CRISPR therapies have made significant clinical strides since we first wrote this blog post... no longer an accurate descriptor; progress in clinical therapy is more fitting, so it is time to update...some solid tumors are currently in early-phase clinical trials and have shown promising preliminary results...introduction of the cancer-fighting component, CAR. Clinical trials are underway to explore CRISPR’s capacity...for these diseases was sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Data shared from these...

...Nature chemical biology. 2021 May;17(5):509-18. Inoue M. Genetically encoded calcium indicators to probe...Iino M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophysical journal...sensors. Selecting a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins...transients in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to... Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor Protocols. ...Measuring calcium signaling using genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;...studies, you can start by asking ‘What is the biological phenomena I want to observe?’ This information...

... capabilities. Specifically this includes primer directed isothermal RNA amplification, new toehold switch...portable quantification of diagnostic results.  Our approach now detects samples at clinically relevant...global strains of the Zika virus (Asian-, African- and American-lineages). Strain discrimination is especially...current outbreak because of the neurological symptoms associated with the American-lineage of the virus. For...populated regions of South, Central, and North America. Diagnostics will play an important role in helping...outbreak was reported, and we felt that, given the critical need for low-cost, field-based diagnostics, our... journal Cell just six weeks later. The Zika publication timeline I think we can speak for all the authors...

...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...that have been genetically modified to either limit or completely eliminate their replicative ability. The...Viral Vectors 101: Viral Applications Viral Vectors 101: Viruses as Biological Tools Viral Vectors 101:...cell. The cell then uses its own machinery to replicate the viral genome and proteins. This is, for the...

...understanding of complex biological processes. Many tools exist to perturb biological function in a direct...use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired...individually engineered and empirically evaluated for their gene silencing efficacy, thereby requiring a substantial...limitations of any technique in the context of the biological question. For example, while gene knockouts have...makes traditional knockouts unattractive from a practical perspective. Therefore, techniques that function...degradation, blocking translation, or chromatin modifications of complementary gene sequences (Figure 1). ...silencing can be easily induced by exogenous application of long or short dsRNA. However, it is important...

.... The overlap here is that it is unethical to use scientifically unproven technologies to edit a human...human clinical trials. This model also demonstrates that when we are at the point of scientifically rigorous...throughout the history of medical research, there have always been people who unethically experiment on humans...Cas9 genome editing is not far enough along, scientifically, to be doing in humans; We have not had a broad... recently published a report on the social and ethical issues of genome editing and human reproduction...with healthy mitochondria, with the intent to eradicate mitochondrial diseases. The key success of the...investigate the scientific possibility, safety, and efficacy of the mtDNA transfer options being investigated...

...cell lines (European, East Asian, Indigenous American, Africa, or South Asian) used in the screens based...Sean and Aaron knew that their study had significant statistical limitations, and expected to find only ...kind of biological framework, suggested to them that their findings may be the result of a technical artifact...Biomedical research has a long and well-documented history of racial bias, which still impacts and influences...Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry associated dependencies...Seeking an explanation  First, they looked for a biological explanation, a relationship between the genetic...machinery where to cut.      Fig. 2: Heatmap indicating loci where SNPs reside on sgRNA target sequences...

...next logical step after technical mastery. However, being a great scientist doesn’t automatically translate...cohesive team environment. Neglecting communication skillsClear communication is the backbone of effective management...transition While a PhD equips you with exceptional technical skills, transitioning into management often means...stepping into unfamiliar territory. Unlike solving technical problems in the lab, management involves a different...time, I discovered that I not only enjoyed the technical aspects of my work but also found fulfilment in...your team's—can lead to frustration. Clinging to technical tasksDelegation can feel unnatural for those accustomed...myself to focus on my core responsibilities, which typically revolved around the broader vision rather than...

...weather befitting of the ominous political climate in which American scientists find themselves today,...we spend years being taught to communicate in a very specific, technical manner, so much so that we often...also a science communicator, UBC Public Scholar, and a member of the Vancouver SciCATs. Follow her on ...March for Science from researchers all over North America. I hope that these perspectives, the optimism they...Alina Chan is a postdoctoral researcher at Harvard Medical School working on human artificial chromosomes....election.   Even as we marched in solidarity with our American neighbours, the fight is far from over here at...April because devaluing science has alarming implications for our public policies—many of which impact...

...the IPI Glypican Collection here! Figure 3: General glypican domain structure. Glypicans are proteoglycans...recombinant antibodies targeting Glypicans By Ashley Waldron Glypicans are important modulators of cell...AAV injection at 4 weeks. Cortical layers (L1-6) and scale bar are indicated. B) Specificity, as percentage...Here's what you'll find in this post: Glutamate indicators with increased sensitivity and tailored deactivation...lentiviral backbones New recombinant antibodies for Glypicans DNA editing with STITCHR Dynamic protein labeling...viral preps targeting interneurons Glutamate indicators with increased sensitivity and tailored deactivation...and iGluSnFR4s (slow deactivation). Both have significantly improved brightness, kinetics, and sensitivity...

...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry...website highlights a variation of your application, a publication successfully used it in a similar way...exciting new project aimed at understanding how a critical cellular pathway is regulated. You’ve read all...choosing an appropriate antibody first look at the application-specific data provided on the manufacturer’s ... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...the most similar to yours. Take a look at the publications cited on the manufacturer’s website, which may...plan to. To secondary or not to secondary?  The application will also determine whether you should use an...

...regular part of basic biological research as well as clinical therapy. These biological tools are useful because...being engineered as biological tools, thought was given on how to keep the replication of virus and its ...the component that physically integrates into the host genome, the virus is replication incompetent, meaning...envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third plasmid...meaning it can’t replicate and continue the infection cycle once inside the host. While the virus can still... this safety feature prevents the virus from replicating and spreading in non-target cells, including ....    Viral cargo-specific safety   After replication competency, the second concern with virus safety...

...in PubMed and choose the appropriate publication. This publication will be listed at the top of the final... with publication details or permission to distribute, or Addgene finds your journal publication describing... "Published plasmids" or "Unpublished or pre-publication plasmids" and set a desired distribution status...quickly associate your plasmids with the original publication where you described them. Search for your article...plasmids. You can submit "Unpublished or pre-publication plasmids": Unpublished plasmids or plasmids that...accepted. Addgene also encourages submitting pre-publication plasmids so they can be available when the paper...preprint submission, we are happy to update the publication information once the manuscript is published ...

...Finding an antibody that works for your application, specifically detects your protein, is species compatible...(stay tuned for our affinity purification tag blog!).   Applications of epitope tags   The...protein purification is not ideal with Myc, as low pH conditions are required during the purification, which...and have been widely used across species and applications. In this blog, we will review the most common...that are bound by antibodies for the purpose of purification or detection of the tagged protein. These peptides...not exist, or they don’t exist for a specific application, or they exist but are unreliable for a variety...almost certainly already been optimized for your application. Depending on the tag, directly imaging or tracking...

...of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites...homologous recombination. It allows for direct modification of DNA within E. coli and is independent of ...engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable...however, only Beta is required when generating a modification with an ssDNA substrate.  Gam: Gam prevents ...insert site (Figure 2, top).  These primers are typically ~70 nucleotides long (20 nucleotides that anneal...regions flanking the target site).  The best applications for dsDNA inserts include: large insertions ...fragments, such as gene replacements and tags.  The typical frequency of recombinants is 1 positive clone out...

...2012) scFv Antibody: Principles and Clinical Application. Clinical and Developmental Immunology 2012:1...therapeutic applications where longer retention times often increase therapeutic efficacy (Ahmed et al...scFvs are often genetically fused to other proteins, which has led to their application for basic and translational...cheaper to generate since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to... translational research.  Basic research applications of scFvs: The SunTag system uses an scFv to amplify...

.... Lajoie, Marc J., et al. "Genomically recoded organisms expand biological functions." Science 342.6156...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...phosphorylation events on the activity of a given protein? Classical methods for studying protein phosphorylation A...steric and charge-based nature of the phosphoryl-modification; they are both much smaller, and carry only ...thus cannot reflect the true state of protein modification within the context of its appropriate regulation...plasmid. This includes 4 copies of a tRNA that specifically recognizes amber stop codons (TGA) to encode...effects on cell growth. The C321.ΔA strain was genomically recoded to eliminate all 321 amber stop codons...

...ASAP5, a faster and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5...responsivity in screening ASAP3 variants. B) Electrical and optical recordings of spontaneous activity in human...protein-coupled receptors (GPCRs) are critical components of many physiological pathways and represent the largest...by endogenous GPCR. Example applications include large-scale pharmacological profiling or interrogation...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration... an enhanced pU6cm promoter, viral replicon amplification of cargo, and heat treatment.  Figure...Arabidopsis, suggesting the improvements may be broadly applicable to dicot species. In addition, they generated...

...Foundation, an organization working to advance biological engineering openly so as to benefit all people...Initiative. Endy's team made news in March with a publication in Science describing the development of “transcriptors...with the BioBricks Foundation to distribute the physical DNA samples, for when that's the preferred mode...parts must flow.' People must be able to use genetically encoded functions - the words in our language...operating system for engineering an entire cell. Typically when people use plasmids, they are trying to do...the entirety of a cell. That's a big long-term technical challenge. We have a strategy that organizes how...work, and it begins with the central dogma - replication of DNA, transcription of DNA to make RNA, translation...

...COVID-19 cases each week. Statistical Modeling Note -- If statistical modeling isn’t up your alley,...breaking the statistical independence assumption of the Poisson process. This modification also eases the...learning and statistical methods for fundamental scientific discovery and high-impact applications.       Alvin... of Chemistry and Chemical Biology at Harvard University; and Howard Hughes Medical Institute Investigator...months, pausing or terminating employment for a significant fraction of the workforce. Wet lab scientists...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...14%). Spikes in plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests...