...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...aims and approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important ...normally one figure and can be negative and/or even replicated studies to show reproducible results. This a ...referenced. In addition the data published in microPublications is curated and added to other databases including...including wormbase and flybase. microPublication is run and maintained by members of the worm community...introducing point mutations that was published in a microPublication (Schwartz and Jorgensen, 2018).  These are...

...post-translational modifications, quantify protein levels relative to standards, detect the cellular location of proteins...pull-down assays.  Westerns are divided into two categories, native and denaturing. In a native western, ...matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper. Pretty much...search, first pay attention to the validated applications listed on the vendor’s website and choose an...western and vice versa.  In addition to validated applications, pay close attention to the controls listed ...and incubation times. If the vendor includes publications that cited the antibody, consider reviewing ... the materials and methods sections of those publications.  Finally, always verify that the chosen antibody...

... its nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being...characterizing the 4D nucleome. Future modifications and applications Ma et al. developed this technique ...have major applications to cancer, for example, the visualization of chromosomal translocations or chromosomal...plasmids from this publication at Addgene. A protein-tagging system for signal amplification in gene expression...technique published every week! One of the newest applications is a colorful system that uses fluorescently...Feb 23. Pubmed. Find the plasmids from this publication at Addgene. Dynamic imaging of genomic loci...Oct 9. Pubmed. Find the plasmids from this publication at Addgene. ...

...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...KCRs), from a fungus-like protist Hyphochytrium catenoides (Govorunova et al. 2022). The authors show that...

...into this category describe how the user is expected to interact with the software. This category is important... CRISPR software submissions. With few prior publications to go on, a submitted manuscript could only ... solutions that facilitate most experimental applications in CRISPR engineering. When compared to that...easy to fault the lack of features in a single application. The question is then, should we use all tools...1 Jan 2013, no longer available), is a simple catalogue of reported sgRNA designs. The second database...novo sgRNA design, I again separate it into two categories. On the one hand we have software tools that ...based on your project needs. There are 8 major categories found in the table, describing everything from...

...addgene.org Addgene's Antibody Purification Protocol Addgene's Video Antibody Purification Protocol Addgene's Antibody...cells can perform human-like post-translational modifications that bacterial and most yeast cells cannot. ...production system may depend on what downstream applications you are planning, cost, and the amount of protein...secretes fewer of its own proteins, making the purification process simpler. Due to its high yields, E. ...production (Schwarz et. al., 2020). Harvest and purification At its core, harvesting recombinant antibodies...your conjugate and conjugation chemistry.  Quantification Once you’ve collected your antibody in the ...accurate concentration to give your downstream applications the best chance at being successful.  Using ...

... inducible translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins...technology, which uses locus-tagging and affinity purification, in 2009. However, iChIP requires insertion ... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic...describe this technology and some of its potential applications? Fujii: We generated a series of retroviral ... discoveries in biology. Therefore, my lab is dedicated to development of new biological technologies....interference-PCR (ORNi-PCR) method to suppress PCR amplification of specific DNA sequences. Thus, we've made ...the peer-review system of journals and grant applications, for example. However, at the same time, it'...

...genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue...After self-excision, the resulting loxP scar is located in this synthetic intron, and only the fluorescent...use and highly efficient for C. elegans genome modification. Figure 2: Schematic from Dickinson et...permission from the Genetics Society of America. Applications of this system Dickinson et al. have developed...system could be used to make many other genome modifications, including targeted mutations. SEC-based systems... in cell culture. In addition to CRISPR/Cas9 applications, SEC selection could replace other, more restrictive... of print] PubMed. Find plasmids from this publication at Addgene. Other strategies for CRISPR/Cas9...

...the rest of the genome and (2) the sequence is located just upstream of a Protospacer Adjacent Motif (...editing is most efficient when target sites are located in close proximity to the region to be edited. ... that were able to cleave target DNA sequences located upstream of either NGA or NGC PAM sequences (Kleinstiver...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...VRER variants is similar to wild-type SpCas9, indicating that the variants are likely just as selective...to dramatically increase the number of CRISPR applications. The first non-Cas9 CRISPR protein adapted for...doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim E, Koo T, Park SW, Kim D, Kim...

...playing a card game called “Magic”. There were complicated rules that governed how this game was played.... as I try being compelling, interactive, and application-driven to boost curiosity, I have never experienced...strategies work. Find Addgene Blog posts about other educational tools Playing BUILD YOUR PLASMID BUILD YOUR PLASMID... cards (Promoters, Inserts, Tags, Origins of Replication, Resistance gene and Selection Markers) and one...building, collaborative critical thinking, and communication among others. They additionally had to think...Who knows, perhaps the ideas spurred by this “gamification” will lead to new cures for disease someday!... each a variety of subjects at many different education levels. If you modify BUILD YOUR PLASMID or generate...

...function. You can also click one location in the plasmid and then a second location to select the sequence between...two separate locations in the linear sequence selects the sequence between the two locations and sends it...table includes feature names, locations, sizes, colors used to indicate them on the map, direction (if...promoters, origins, tags, and certain ORFs. Their sophisticated detection algorithm easily and precisely identifies...restriction enzyme annotations throughout the map at locations better aligned to their precise positions within...between the two locations and send it to the clipboard for copy and paste functionality. Sequence ...sequence. The table includes enzyme names, the locations where the enzymes will cut (in basepairs), and...

...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give... protein interactions, including epigenetic modifications. But it's not an approach for the faint of heart...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...to the selective nature of the cutting. While sonication is usually the preferred method, enzymatic digestion...concentration.  Protein Capture After crosslinking and sonication or enzymatic digestion, you’re ready to incubate...crosslinking. Finally, purify your DNA with a PCR purification kit or with a phenol-chloroform extraction.  ...

...labeling can be subdivided into two categories, depending on the location of the targeted markers. Some commercially...proteins, such as receptors, or post-translational modifications on those proteins, like glycosylation patterns...Depending on the research question and downstream application, one or several of those techniques can be used...your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those ...labeling Oftentimes, a marker of interest is not located on the cell membrane but inside the cell. Due to...but preserving the cellular state (think of mummification). Permeabilization, as the name suggests, perforates...the nucleus intact. If markers of interest are located inside the nucleus, a different fix/perm kit is...

...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...characteristics, performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova...information on NIR FPs, their properties and applications. At the end, there is a Table that summarizes...photoreceptors incorporating biliverdin IXα (BV) autocatalytically. Fortunately for probe developers and users...vivo probes, due to low autofluorescence, low scattering, and deep penetration of red-shifted light. The...their enhanced versions named emiRFPs in your applications.  There is a whole set of spectrally distinct...of Medicine. Her expertise is development and applications of fluorescent proteins, genetically encoded...

...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including... carboxylase and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids...this highly specific post-translational protein modification. Most organisms have less than five enzymes ...uses of biotinylation in the lab are protein purification and immunoprecipitation experiments. Biotinylated...sites found on avidin are also ideal for signal amplification during these experiments. During detection, ...bound to your protein of interest leading to amplification of the reporter signal and easier detection....

...current CRISPR research focuses on its potential applications for human medicine (Waltz, 2016), the potential...engineering, on the other hand, allows for targeted modification of known or suspected genes that regulate a ...model organisms like Arabidopsis and Medicago truncatula and several crop species including potato, corn...engineering using CRISPR, highlight the specific modifications to CRISPR machinery that allow for the use of...CRISPR principles). However, plant-specific modifications to commonly used CRISPR plasmids are necessary...selected the correct CRISPR components for your application, it is time to deliver these components to your...detergent, Polyethylene Glycol (PEG), although the application of this approach is limited to protoplast cells...

...mismatches on modification by AsCpf1 (red) or LbCpf1 (blue). Mismatch positions are indicated below each ...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...compared the ratio of total off-target to on-target modification for AsCpf1 and LbCpf1, and found that both orthologs...the PAM sequence. Many research and clinical applications require HDR over NHEJ, so comparing HDR efficiency...PubMed PMID: 27272384 Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et ...PubMed PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh...

...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...expected fragments for that plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1:...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...observed to exist in various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization...other methods below should help in multimer identification.   Fig. 2: Gel image of Plasmid 61564...within a plasmid and, therefore, allow for the identification of plasmid multimers. Capillary Gel Electrophoresis...multimerization. One study by Bacolla et al. (2011) indicated that strain HB101 produced the highest yield of...

...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...that fall into this category include performing experiments with too few replicates leading to suspect ...these positive results will be published. The publication record will then show that the tested hypothesis...reproducible publishing practices. One example is the publication of peer reviews so we can see how and why papers... also means that you’re more likely to get a publication (for better or worse, the currency for success...review of experimental design as part of the publication process (Nosek et al 2018). In this ideal situation...

... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...tool with magnets would enable new research and applications that don’t yet exist, but it wasn’t clear that...changes in response to a ~25 mT magnet. Gray regions indicate times when the magnetic field is on. Image reproduced..., in response to a ~10 mT magnet. Gray regions indicate times when the magnetic field is on. Image reproduced... shows a wealth of potential, and while many applications are still yet to be demonstrated (or invented...

...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes.  ... steps of AAV production, iodixanol gradient purification, and AAV titration by qPCR.   Pro tip! An optional...al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from ...Benyamini et al., 2023) to save time and cost.  Purification To purify your rAAV from your supernatant, you...recommend using a Tris-based lysis buffer and sonication.  The obtained virions from cells and supernatant...

... gene’s transcription. Sometimes enhancers are located thousands of base pairs away from the gene they...created a dCas9-based CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The ...could only detect chromatin interactions at one location in the genome at a time (Liu et al., 2017). Recently...at once (Liu et al., 2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original...biotin ligase a gRNA(s) targeting a single genomic location of interest  When these three components are ...together in terms of where they are physically located. DNA is then sheared into pieces small enough to... original CAPTURE and CAPTURE 2.0 methods.   Applications of CAPTURE 2.0 CAPTURE 2.0’s increased rate ...

... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your...is a graduate student group with the goal of communicating science to the general public and training the... the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...Pint" event is the best way to get a Science Communication group started. It's as simple as getting people...

...my opinion, it was more important to communicate the applications of CRISPR over the details of how it...m a father of three, a scientist and science communicator, and I strive to be a volunteer and contribute...recent experiences. STEM in elementary schools - catching them early I’m fortunate to live in a town with...unlike elementary schools, middle schools have dedicated science teachers. I tried contacting our school...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...great feedback from teachers and other science communicators.  What can CRISPR do? I spent more time on...children and you want to make sure their STEM education is supplemented. A 2009 study by the NSF showed...

...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required...development. David Liu’s Lab has demonstrated the use of cationic lipid-mediated methods to deliver Cas9-gRNA RNPs... of eventually using this method for in vivo applications. This technique utilizes Cas9 proteins harboring...CRISPR experiments can reduce many potential complications, there are additional factors to take into account...

...If you use a kit for DNA purification or if you use a DIY purification protocol, you might have noticed... mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade...degradation during plasmid DNA extraction and purification from E. coli is the presence of the protein ...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been...chelating agents that can be introduced in the purification protocol to remove endonucleases activity. For...controls the pH, while the EDTA chelates any divalent cations like Mg2+, preventing the activity of DNase. While...do with these samples. This is because divalent cations are essential cofactors for many tools used in ...

...logistical barriers, allowing people to access a wide catalog of plasmids from all over the world. This idea,...into the accessible. Physical access Addgene is located in the USA, but our materials are available to ... items to countries with a distributor. Arrows indicate the year we entered into an agreement with a distributor...accessibility to researchers around the globe. Addgene’s educational resources As Addgene built out our scientific...guides, protocols, and collections of 2023. Educational Resource Top Five by Views Guides CRISPR...Systems AAV Plasmids Blogs Origin of Replication What is a Plasmid? How to Design Your gRNA for...countries. In Table 1, you can see our most popular educational resources for each type of resource we offer....

...the other to guess a category by listing off things that fall into that category? Okay, let’s play. I’...’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot ...concentration of viral genomes (by qPCR or other DNA quantification methods - see below), since each viral particle...value, physical titers don’t necessarily give any indication of the infectious titer. The infectious titer...affected by many experimental factors. To list some complicating factors for quantifying AAV: qPCR efficiency...optimized our assay in a few ways:  Absolute quantification by qPCR requires generating a standard curve...plasmid standards. We validate our absolute quantification by using the universal AAV Reference Standard...

...advancements. Consultants can help these teams develop communication strategies, or understand, and publish on the...success. Or the client might need help with the intricate task of designing a strategic vision for a portfolio...investments over the next 3, 5, or 10 years. Complicating these challenges is the fact that they occur...chemotherapies, or developing innovative mobile phone applications and services that help diabetic patients manage...knowledge from previous consulting experiences, educational training, and conferences, and synthesizing this...that gives the client a defined path forward. Communication is key as ideas must be relayed quickly both...clients and experts. Hence, active honing of communication skills is a must to be successful in this field...

...cellular impediments. FRET has been used in many applications, including drug discovery by big pharma (i.e... acceptor doesn’t need to go to the same exact location as the donor. As long as some of the acceptor ...C-terminus of your target protein, but even these modifications can affect the structure or function of certain... close to the donor, faulty delivery to other locations in the cell are not a problem, as long as they...The poly-histidine tag often used for protein purification is a reliable FRET acceptor labeling site when... way as NTA-sepharose beads used for protein purification: the NTA binds to the His tag on your protein... used to fluorescently label antibodies, and purification is a snap using thin layer chromatography (Fessenden...

...plasmids and their potential educational uses. We believe that biology education is something that should ... expensive tiny amounts of reagents are, how complicated (and again, expensive) the different machines...desire to create a set of low-cost, easy-to-use educational biology activities – that any school could afford...proteins without the need to handle a living, replicating organism. Furthermore, cell free systems can ...in adapting your research ideas into hands-on educational activities using the BioBits platform, please...BioBits™ Explorer: A modular synthetic biology education kit." Science Advances 4.8 (2018): eaat5105. PubMed...BioBits™ Bright: A fluorescent synthetic biology education kit." Science Advances 4.8 (2018): eaat5107. PubMed...

...overall. Communication is the issue Much of the problem can be attributed to communication, and in particular...particular communication about experimental methods and approaches. It’s very easy to think that the methods...that they were doing something differently. Communication comes in many forms—from transparency about ...them troubleshoot and understand. All of this communication and sharing fits together to make for a transparent...for pushing for more transparency in science communication falls on the shoulders of journals and publishers...can feel like yet another hurdle in front of publication when all they really want to do is get the paper...we take our responsibility to push for clear communication of science very seriously. That’s why at Cell...

... a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for... their bacterial counterparts, including how replication occurs and whether selection is necessary for...the nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted ...always mission critical to make sure the plasmid replicates in the cell, as many of these experiments are...expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr virus...antigen (293E or 293T cells), allow for episomal amplification of plasmids containing the viral EBV or SV40...contain an ORI since the integrated DNA will be replicated as part of the genome. Because the foreign DNA...

...check its subcellular location and solubility. If it’s membrane-bound or located in a subcellular compartment...nuclear- or DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins ...will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant wells...membrane into the psi membrane, that’s a good indication that the p membrane pores are too large.  Transfer...likely technical. White circles, for example, could indicate that you had air trapped between the membrane ...during the transfer, while dark splotches could indicate dirty equipment, degraded or improperly mixed ...using an antibody validated for your protein and application of interest or validating an antibody yourself...

...led to their application for basic and translational research.  Basic research applications of scFvs: ...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...from blood can be a drawback for therapeutic applications where longer retention times often increase ...since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies, there are many...2019).Many of the scFvs designed for oncology applications are reformatted as bispecific molecules which... autoimmune, and/or inflammatory therapeutic applications. Antibody fragments such as scFvs are also used...

...resistance cassette, and the R6K origin of replication to allow replication only in hosts carrying the bacteriophage...chromosome. These methods roughly fall into two categories: those that rely on exogenous enzymes and those...Lambda Red recombineering, which uses enzymes domesticated from the Lambda bacteriophage, is arguably the...enzymes can also accomplish the desired genomic modification. This technique is frequently called allelic...recipient of interest. Because pDS132 cannot be replicated in the recipient, the only antibiotic resistant...exchange. To do this, we performed the following modifications to pDS132 (Philippe et al., 2004): Replaced...Lazarus et al., 2019). Additionally, to facilitate modification of strains where conjugation may be difficult...

...recombinase polymerase amplification (RPA) using a commercially available amplification kit. In this case,... RNA is extracted and subject to isothermal amplification by primers that target the E and the N2 gene...reverse transcriptase to produce cDNA. Isothermal amplification is done at one constant temperature, eliminating...rapid detection.  Highlights: Cas13a is not catalytically active in the presence of two or more mismatches...that exhibit fluorescence upon cleavage. Upon amplification by the RPA primers, the Cas12a-crRNA binds to... SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect as low as 1.2 copies DNA..., field-deployable nucleobase detection and identification using FnCas9 (FELUDA) Sensitivity: – 110 femtomolar...

...conflict is not always what was communicated but how it was communicated. The next step is to come up with...of lapsed communication. Making some assumptions about how something should be communicated is necessary...skill in and of itself. Conflict can also be categorized as either “personal” or “substantive”. A personality-based...traced back to one of a few trouble spots:  Communication (or lack thereof): Information sharing gets ...organization gets, and creating new avenues of communication can create as many problems as it can solve....meeting? Different teams frequently prefer to communicate in different ways, so the source of the conflict...with a viable process that will avoid such miscommunications in the future. Assumptions: Making incorrect...

...pre-treatment levels due to the viral reservoirs located in latently infected cells. Although ART-treated...toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. Cutting HIV...create mutations in genes essential to HIV-1 replication. However, this approach could permit some viral...along with Cas9 in the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed... HIV-1 could escape from CRISPR/Cas9-induced modification targeting either the LTRs or essential genes...genes. Many of these escape mutations were located near the Cas9 cut site, leading Wang et al. to conclude...lessons learned in pursuing one technological application can benefit many others and accelerate the pace...

...ligation using SpyLigase Building off their SpyTag/SpyCatcher system for protein-peptide locking, Mark Howarth... a critical role in development and are often implicated in disease progression. One of the major challenges...challenges in signaling research is the inability to replicate the spatio-temporal precision with which signaling... of complex biological systems requires both a catalog of molecular players and contextualized knowledge...the most abundant forms of post-translational modifications in cells and research into its many roles in...Alex Schier's lab, designed for expression and purification of Cas9 protein in Rosetta E. coli. New Lentiviral...FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically inactive Cas9 DNA-binding domain in mammalian...