... or pharmacological manipulations on cells, and has potential use for a wide range of applications in ...platform at Addgene. Braselmann E, et al. Nature Chemical Biology. 2018 PubMed PMID: 30061719. New tools...expression vector (pRSETB). Fabritius A, et al. Cell Chemical Biology 2018. PubMed: PMID 30220597. ACE system...versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9 cleavage activity ...plasmid that allows for rapid fluorescence-based quantification of base editors that utilize dCas9 fused to... The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry fluorescence is...frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply involves tracking...

...type; however, their usage has been limited by technical problems. For instance, it’s much more difficult...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...potential for off-target effects. Third, protein purification (once a protocol has been established) may be...

...Harvard Medical School and the University of Toronto. He is an author of 20+ scientific publications and ...quick turn-around time of Sanger sequencing have historically matched the needs of most molecular biology ...smaller fragments that are then barcoded for identification. This mixture of barcoded fragments is known... a collection of 96 plasmids on a plate) will typically have a wide range of input quantities. This variation...samples. As mentioned above, one of the primary technical benefits of plexWell™ is that normalized libraries... proprietary chemistry). This turns out to be critically important for getting adequate sequencing depth...computational biologist with a passion for solving critical sequencing problems. He received his PhD in Bioinformatics...

...MSDS for a lab chemical of choice. Now, I can’t guarantee every reagent in your chemical closet is in this...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder...enzyme sequence resource is universally useful. 4. Chemical Safety Data Sheets by ThatsMyStapler Safety always...experiments. Hence, I’m including this MSDS app. The Chemical Safety Data Sheets app has a search function that... if you get quizzed randomly about a hazardous chemical and can’t find your lab’s MSDS book, this is a...AMINO is a very simple app that packs important biological information, amino acid abbreviations, and structures...

...these proteins are FDA-approved or in clinical trials or preclinical studies already. We are following up...expand to other cell types, including more physiologically-relevant systems, to catch all the important... we found in our map have an impact on virus replication. While we and our collaborators are starting ... Nsp3 and Nsp16, and includes an additional catalytically dead version of the viral protease, Nsp5. Our... 2xStrep-tag for easy detection and affinity purification. All but one are in a pLVX vector background...coronavirus, one FedEx package at a time Since the publication of our paper, our group has been inundated with...Eckhardt and Melanie Brewer are on the Science Communications Team at QBI UCSF. They are particularly interested...

...agreement on the transfer of biological materials in 1995: the UBMTA. Because biological materials are widely ...many different flavors of MTAs, but the Uniform Biological Material Transfer Agreement (UBMTA) is one of...MTA for that plasmid facilitated by Addgene — basically, instead of having to sign individual MTAs for...things being organized, tracked, and clearly communicated. And that’s where MTAs, and similar agreements...agreements allow for centralized tracking when biological reagents are shared — both at what time and with... options for the terms you want to share your biological materials under! While most plasmids at Addgene...portal automates a lot of the process, from notifications and emails to generating the UBMTA link for ...

...ll be talking about the ins and outs of this classification system, including its history, biology, and...repeatable phenomenon, Gram began using it as a classification system. Eventually, any bacteria that retained...gramstain.html Smith, A. & Hussey, M. Gram Stain Protocols. American Society for Microbiology (2005). https://asm.org... or pink. Purpose Gram staining can be used in medical settings to determine the general type of bacteria...permeable to different classes of antibiotics. The classification system is also useful in a laboratory setting...bacteria type. The gram positive and negative classification system is incredibly helpful in visualizing...stepping into a lab.   Anna Kamens is a science communication summer intern at Addgene and a rising third-...

...Technology and its Application in Genetic Diseases: A Review.” Current pharmaceutical biotechnology. https...2020.  “CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future.” Front. Oncol...several treatments are currently being tested in clinical trials. It is speeding the process of the discovery...the technology in different labs for different applications.” Li’s team noted that CRISPR/Cas9 has been ...cleavage sites are repaired.” Those insights will be critical for scientists’ ability to influence editing efficiency...with size limitations. In addition, non-viral applications are also possible through the use of nanoparticles...

... and 15/17 morphologically aligned. Documentation of the network that initiates biological reprogramming...unique barcodes for each TF help streamline identification. While the Zhang lab controlled their multiplicity...single TF was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing...processes. For help with library transformation and amplification, refer to the Zhang lab’s previously published...published protocol and its relevant modifications on the pooled library webpage. Disease models Good disease...generate, in part because of difficulty with replicating the specific dysregulation in the disease. For...Addgene.org Plasmid Pooled Libraries Pooled library amplification Zhang Lab CRISPR Plasmids The Human TFome Library...

...mentor (if you have one) to communicate your plan as well. Making logistical and funding arrangements Visiting...the host university does not hire or pay them (typically). A visiting scientist is distinct from equipment...the scientist to the new institute where their typical workdays will be carried out until the end of the...equipment! Preparing for your visit   Design and communicate a research plan Your time at the new research... and you are set up for success. If you are communicating with a professor or institute, ask if you will...expect to generate data that will contribute to a publication? A grant? A patent? It is never too early to ...Resources Resources on the Addgene blog Fostering communication in remote collaborations Transferable Skills...

...researcher and science communicator.  Update (7/17/2018): Check out Fatima's recent publication on her HMO work...protein-protein interactions. Beyond these common applications, researchers are finding new ways to apply fluorescent...Zhou and Fatima Enam. There were many excellent applicants but Xin and Fatima stood out for their creativity...utility of these FLiPs in a variety of research applications and particularly notes their ability to be precisely...known to bind to the HMO of interest. Fatima specifically proposes using this sensor to detect HMOs found...endeavors, Fatima hopes to practice her science communication skills throughout her training and wants to ...work in Cell Chemical Biology! Additional Resources on the Addgene Blog Learn More about Photosactivatable...

... Connie Cepko (Harvard Medical School), and Dr. George Church (Harvard Medical School) for taking the ...does one stand out amongst a pool of successful applicants and get chosen to work in his or her lab of choice...environment? Does the group expect each other to be physically present at certain times during the day? We asked... next.” Connie Cepko looks for people who are dedicated and interested in science. “Postdoc hires are ...history in science. Potential graduate students typically go through a rotation, which reveals their working...job satisfaction (especially for scientists and technical people) is linked to engagement and access to ... "chaotic" and offer less micromanaging, they typically offer more flexibility in choosing direction of...

...systems are available at Addgene! Chemically inducible promoters Chemically regulated promoters are among ...sensitive expression systems are typically less leaky than chemically induced promoters; they show near-zero...transcription is turned ON. Types of inducible promoters Chemical agents, temperature, and light are all examples...prokaryotic promoter often used for bacterial protein purification. When arabinose is absent, regulatory protein...decreasing promoter leakiness.   Other examples of chemically induced promoters include positive inducible ... promoters only work in prokaryotic cells and typically only in the same or closely related species from...

...emphasizing the importance of critical thinking, persistence, teamwork, and communication. And as a program focused...BioBuilderClub teams’ biological engineering designs and ideas ranged from a biological breathalyzer to pesticide... Dr. Natalie Kuldell from MIT’s Department of Biological Engineering. Built primarily from her 20 years...experiential learning. “Students may forget the technical details they hear in their classes,” she said,...and teachers to apply engineering principles to biological systems. Today, what started in Cambridge with...Apprenticeship, preparing local (Massachusetts), economically disadvantaged students for paid summer internships...

...cost-prohibitive for many labs, and are often impractical for small projects. Most researchers instead ...involves splitting up a sample into thousands of physically isolated partitions (such as droplets or wells...Since there are many thousands of partitions, statistical modelling can be used to determine the actual..., does not require standard curves, and can theoretically detect a single target molecule in a sample....a gene of interest is one of the most common applications of precision genome editing, the remainder of...single molecule resolution of digital PCR allows classification of individual target molecules based on their...single wells of a 96-well plate without prior quantification. Comparatively, mismatch assays require between...

...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify and study...Research Assistant in Arlene Sharpe’s lab at Harvard Medical School.   More resources on the Addgene blog Antibodies...addgene.org Addgene's Antibody Guide rAb Affinity Purification Protocol Video rAbs in the Addgene repository...

...Following up its 2018 iGEM debut - centered around a prototypical methane biosensor - IISER Bhopal is back in ...psychrophilic enzymes for milk processing, fruit juice clarification, meat tendering, and oligosaccharide synthesis...enzymes implies that even a minute quantity can significantly reduce the operational time for a given process... from their native conditions of ~0°C, which drastically affects their stable folding and efficient functioning...chaperones (the Cpn 10/60 isolated from Oleispira antarctica, and the P. haloplanktis Trigger Factor), that...Additionally, we’re also working towards widening the applicability of our strategy using the pMBP-LbCas12a and ...cultivating a nuanced understanding of the societal ramifications of laboratory science, and how it may best be...

... microscopy. This application of AHA labeling is known as fluorescence noncanonical amino acid tagging...PhD students at the Max Planck Institute for Biophysical Chemistry, Göttingen in Germany. I attended this... symposium seeking inspiration to resolve the technical hurtle of monitoring protein turnover of specific...technique applies metabolic labeling using a noncanonical amino acid, azidohomoalanine (AHA). In the technique...DNA strands take part in rolling circle DNA amplification. The amplified DNA segment is labeled with fluorescent...directed toward AHA. Now the rolling circle amplification could only take place if the two antibodies ...interested in neurobiology.  She is passionate about communicating science to all audiences. Right now, she is ...

...and success. Customer and technical support Our customer and technical service brands continue to ...to keep up with the demand with excellence and dedication. We don’t have worry or wonder if providing...projects allows us to support more new projects. Diversification is happening. The Addgene team will have some... We score ~99.9% helpful and the customer and technical service teams put 100% effort into helping our...we might do. I am grateful every day for their dedication, flexibility, innovative thinking, and openness...Addgenies support a high collaboration, high communication, high efficiency, high quality, low meeting,...about the Addgenies. I never worry about their dedication, excellent intentions, or incredible drive to...

...red fluorescent proteins) allow simultaneous identification of more than one protein of interest. However... species (bottom panel in Fig. 1). Our results indicate that the tag protein may have an important influence...plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to the plasma...However, in some cases the fluorescent signal may be critical for the experiment. Since the fused protein could...fluorescence expressed in the neuropil conceals identification of opsin-expressing cells). For other things...Research Center. Using a combination of in vivo and anatomical techniques, her research aims to gain a deeper...Rodgers L, Wilson IA, Lerner RA, Wigler M (1988) Purification of a RAS-responsive adenylyl cyclase complex...

...repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time ..., 98% of TdTomato expressing cells were GFP+, indicating a robust, but specific system. T-DDOGs also successfully... types of proteins. They started with another biological workhorse, Cre recombinase, which induces recombination...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Tang note that their scaffold system is likely applicable to many types of proteins. For proteins with ...Could Cas9-DOG be developed soon to allow genome modification only in a small subset of cells? Other proteins... PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “...

... work around this is to "domesticate" your fragment: PCR-based amplification can be used to create silent...and exciting applications, this system is an incredibly powerful tool for cloning complicated constructs...plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning...restriction sites that may not be amenable to "domestication", you might want to consider using an alternative...design of flanking overhangs. Although there are theoretically 256 distinct flanking sequences, sequences that...high-efficiency step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS...

...undergraduate students (1, 2), but educators face significant challenges in designing suitable projects. In... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...functional MET genes to grow.) Students then use replica plating to determine if expression of MET/Met genes...met deficiencies (manuscript in preparation), indicating that Met protein functions have been largely ... Students who participate in the course show significant gains in both content knowledge and research ... research into undergraduate courses and in communicating science to the public. You can find out more...

...production and subsequent necessary modification of these chemical products. The flexible nature of MoClo... Gate cloning is MoClo. That’s because MoClo specifically refers to the organizational schema described...organization schema MoClo can be broken down into three hierarchical levels: individual DNA parts, transcriptional...together. They can express one or more gene(s), and typically begin with a promoter and end with a terminator...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...microbially or in cell-free systems. These pathways typically involve multiple steps in which a precursor molecule...genotype and phenotype is best described with “it’s complicated.” Applying MoClo Interested in using MoClo? You...

...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...researchers to identify and isolate very specific biological targets. However, as popular and useful antibodies...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...antibodies, creating a potentially unlimited supply of identical antibodies (See Fig 1.). rAbs are a relatively...a research assistant in lab of Zhuhao Wu at the Icahn School of Medicine at Mount Sinai.  References ...

...generated in vitro using synthetic genes that are typically expressed from a plasmid or from an integrated...cells, for antibody production and subsequent purification.  Academic laboratories and companies have already...share, making recombinant antibodies the most practical choice. Hybridomas are much tougher to ship, often... often lack sequence data, and may genetically drift over time. Antibodies made from animals are limited...the ease with which scientists can now improve genetically-encoded research tools, it is shocking that the...Begley CG, Ellis LM (2012) Raise standards for preclinical cancer research. Nature 483:531–533 . https:/...Edition. 2104. Cold Spring Harbor Laboratory Publications, New York. Helma J, Cardoso MC, Muyldermans ...

...very useful in the event that you will not be physically present at your poster at all times, so other... sections should be as concise as possible, communicating only the most important results to your audience...have the advantage of being able to verbally communicate your work, so you don’t need every little detail...readers can navigate your poster and follow the logical progression of your work, and you can accomplish...offers free poster templates, although most are vertical.  Large central box. Another commonly used and...presentation. Portrait. This style displays a poster vertically rather than horizontally. Instead of displaying.... Shapes. In this type of poster, written and graphical information are inserted into larger circles or...

...Lacks - the African American woman whose cervical cancer cells became a mainstain of biological study without...advice on how to dive into new forms of science communication. Click here to listen to the interview with ...fossil rocks and their importance in understanding biological history. 4. Please Don't Kill My Hive Beautifully...implement the next generation science standards and communicate science. Learn more about Tom on his youtube ...

...example, if you’re a virologist presenting at the American Association for Cancer Research Annual Meeting...therapeutic strategies to target it, than you typically would at a microbiology meeting. Create an outline...lab mates, and PI can provide feedback on your technical presentation, but those outside of your field ...don’t fully understand the question, ask for clarification. Having them reframe or elaborate their thought... through the list at the session so you can strategically use your time. Don’t have access to a list of...titles to find the ones you’d like to revisit. Strategically traverse the sea of posters If you come prepared...system, and is interested in a career in science communication.       Resources on the Addgene blog Read other...

...recognized by its antibody without additional chemical modification steps and when the epitope was within the...antibodies for every potential new target and basically impossible if you wanted to distinguish between...C-terminus post-translationally, which turned out to be critical for antibody recognition. As such, the peptide...interest and proteins tagged with it needed to be chemically treated before the antibody could detect them... the N-terminal half of the antigen differs significantly between human and the other species, suggesting... flexible - it could now be used in way more applications.   Fig. 1 Making a Protein Tag (Abridged... of new versions of Myc1-9E10 mentioned above basically involve taking the Myc1-9E10 variable regions ...

... more than 10 modules often rely on two-step hierarchical approaches using different Type IIS restriction...stability, and buffer compatibility have placed practical limits on single- or two-step assemblies. We have...upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding sequence... While T4 DNA Ligase, the mainstay of most biotechnological cloning efforts for over 50 years, prefers...-Crick base pair substrates, it demonstrates significant activity on some mismatch-containing pairings... sequence-dependent ligation efficiency and identification of mismatch-prone pairings. Using these 4 bp...Kucera is a Principal Development Scientist of Applications & Product Development focusing on DNA cloning...

... the pharmaceutical industry for years. They have proven very difficult to target therapeutically due ...al. "Systematic identification of signaling pathways with potential to confer anticancer drug resistance... millions of people die from cancer-related complications each year. Despite these grim statistics, new... cancers, and synthesizing compounds to bind specifically to their mutated residues. For Ras researchers...any of these genes through several different applications such as fluorescent protein fusion studies and...represented, making the results of any such study as clinically relevant as possible. Connecting the research...

...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing...PMID: 26678082. Find the plasmids from this publication at Addgene. 2. Dalvai, Mathieu, et al. (2015...633. PMID: 26456817. Find plasmids from this publication at Addgene. 3. Zhang, Chi, Xiuhua Meng, Xiaolei...

...transformative biological discoveries and life-saving medical breakthroughs and undermine critical research ...Addgene as a trusted partner for archiving their biological materials during funding transitions. Enabling...operating model allows researchers to access authenticated materials at a fraction of the cost of creating...be listed as direct costs from a CRO in grant applications, addressing the new funding structures and indirect...private partners to create additional pathways for critical scientific work to continue.  Moving Forward ...

... citations for recent articles. Of course, a publication in Science or Nature is always nice, but in the... lesser-known journal as it is in a top-tier publication. Accessibility is surely also behind the increase...increase in citations for older publications. (As an interesting aside, Nature recently reported that the...in a solution.) But are citations and journal publications really the best way to measure impact? As that...papers detailing Nobel Prize-winning research typically weren’t among the most cited. And, as scientific...and what it’s made of. Increasingly, journal publications alone aren’t enough. Scientists are sharing ...consider and quantify the impact of scientific publications. Ventures like Impact Story seek to find ways...

... and chemical contaminants that have been used during the purification procedure. Good indicators of DNA... is necessary for bacterial transformation of chemically and electrocompetent cells is minimal (picogram...nm should fall ~1.8 - 2 and give scientists an indication of purity versus protein contaminants. The 260...nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents as guanidine...addition to being free of any nucleic acid and chemical contaminants, these DNA samples must also be entirely...

...immunoprecipitation applications, but none of the traditional tags were suitable for both applications. To prove...from non-vertebrate systems. Preliminary testing indicated that inntags IT5, IT6, and IT10 expressed well...and the chaperone Ydj1, both of which display sophisticated interactions with multiple other proteins. Again...detrimental effects on cell size and growth rate, indicating that they compromised protein-protein interactions...These tags are easy to use in multiple common applications. Due to their limited effects on native protein...particularly in single-molecule or high-throughput applications. Perhaps IT5 and IT6 will even replace our older...

...conventional microscopes and give a similar optical quality (magnification of 140X and 2 micron resolution). I... science communication and outreach - if you'd like to learn more about Science communication check out...listening to talks all day. But nope, not at the American Society for Microbiology 2018 Microbe meeting....biomolecule-based dyes. This technology is widely applicable as bacterial expression of dyes allows dye visualization...Bacterial Genome Engineering Learn about Science Communication using Twitter  Resources on Addgene.org Check...

...Balancing research with medical practice as an MD/PhD. Darrel does 20% clinical work 80% research and was...for Regenerative Medicine at Boston University Medical School. Darrell’s research focuses on the lungs...Center for Regenerative Medicine, and within the biomedical research community. Along these lines, Darrell...Pathology and Laboratory Medicine. He attends in the Medical Intensive Care Unit, the Pulmonary Consultation...the Pulmonary outpatient clinic, all at Boston Medical Center. In addition he conducts basic stem cell...Regenerative Medicine of Boston University and Boston Medical Center. Special thanks to our sound engineer/producer...

...provided here for reference: OpenWetWare Preparing chemically competent cells (protocol)  Preparing electrocompetent...protocol)  Untergasser Lab Preparation of chemically competent cells (protocol) Preparation of electrocompetent...until we are ready to perform transformations. A typical 200mL bacterial culture produces 20mL of competent...transformation protocols below: OpenWetWare Transforming chemically competent cells (protocol)  Electroporation (...protocol)  Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of...being performed at Addgene, this adds up to a significant time savings. You can also save yourself time...plasmid is introduced to bacteria, the bacteria is typically grown in liquid culture without antibiotics to...