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...ITRs), hairpins, or strong terminators. Detailed instructions on other factors to consider when designing...community. This is no small task, however, at a repository that consistently receives around 200 new DNA...are sequencing from the exact same stock that we store and distribute from. Of course, your culture should...overnight cultures for glycerol stock production and storage, we grow a little extra and transfer 100 µl of ...Pages Get Plasmid Backbone Information from Our Vector Database  ...

...ask the depositing laboratory to review the discrepancies. If the depositing laboratory confirms that these...reference sequence or a sequence provided by the depositor. Depositing labs often provide insert sequences... actin promoter, and rabbit beta-globin splice acceptor, contains sequence that is over 80% GC. Some IRES...information about our sequencing results in the Depositor Comments section at the bottom of the plasmid ...hope to make available as part of our OpenBio Repository sometime soon. Stay posted!     References ...

...engineered prime editors to expand their PAM flexibility. Find the engineered prime editors here A lentiviral...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...pubmed.ncbi.nlm.nih.gov/24390935/ Hypercompact genome editor CRISPR-Casɸ By Melina Fan The Doudna Lab adds to...discovery of CRISPR-Casɸ, a hypercompact genome editor from the Biggiephage clade of huge phages. In this... Scientists looking for more compact genome editors due to delivery constraints and scientists seeking...

...Acr) proteins are phage-derived small protein inhibitors of CRISPR-Cas systems that help phages evade ...the best time to shut off Cas activity? Using inhibitor timing experiments, the Doudna lab found that ...also be used to reduce off-target edits of base editors, which convert a nucleotide base pair to another... means they can be delivered in vivo using AAV vectors. Having a way to shut off CRISPR-Cas systems prevents...Maxwell KL, Doudna JA (2017) A Broad-Spectrum Inhibitor of CRISPR-Cas9. Cell 170:1224-1233.e15 . https...Papayannopoulou T, Lieber A (2018) HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR...

...This guest post was contributed by Johnny Kung, Director of New Initiatives for the Personal Genetics Education...dialogue between scientists and communities. As the director of new initiatives at pgEd, I take on a variety...education and government; advising writers and directors in Hollywood; and working directly with community...sticky ethical dilemmas to dark episodes in the history of eugenics and research misconduct. Past ethical... in genetics research, their applications and regulatory policies; keeping tabs on the conversation about...implications of their work. Johnny Kung is the Director of New Initiatives for the Personal Genetics Education...

... While lentiviral vectors are popular gene delivery tools, producing lentivirus, can pose certain challenges...systems such that it no longer requires the viral transactivator, Tat. For more information on the different...of concentration method depends of a variety of factors such as resources available, cost and downstream...determination of the packaging limit of lentiviral vectors. Hum Gene Ther. 2001 Oct 10;12(15):1893-905. PubMed...Tips for titering your lentivirus Visit the viral vector featured topic page Resources on Addgene.org ...Guide Find lentivirus plasmids Browse all viral vectors ...

...biosensor affinity for calcium.”  Primary Vector Type The vector type should describe how your plasmid ...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...plasmids containing the ccdB gene, such as Gateway vectors, choose the ccdB Survival strain. For plasmids ...Distribute pending QC. The name of the Principal Investigator and Organization where your plasmids were first...

...Enhancers are cis regulatory DNA sequences that, when bound by transcription factors, can increase a gene...CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The original CAPTURE method...2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original CAPTURE method The original... three components: dCas9 with an added biotin acceptor site BirA, a biotin ligase a gRNA(s) targeting...patterns in chromatin interactions. The chromatin story told by CAPTURE 2.0 is important, but it’s not the...

... post was contributed by Laura Desrochers, a postdoctoral scientist at AstraZeneca. Why I chose an industry...time working on something extremely risky or exploratory. Getting a job afterwards: Fairly or unfairly...an industry setting. Scientific community and mentorship: As an industry postdoc, you might find yourself...project’s or a team’s, it is possible that your mentor (and even their department) could be laid off while...Desrochers! Laura Desrochers is currently a Postdoctoral Scientist at AstraZeneca. She has a background...blog posts here Find 9 tips for a successful postdoctoral experience Read Joanne Kamen's 8 tactics for...

...My lab's vector of choice is AAV, with nearly every experiment requiring AAV. Before joining my lab, ... a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too hot because...approach gives me a bit of flexibility since I can store the cell lysates at -80 °C for up to 6 months before... completed in one long day, or the virus can be stored at 4 °C and buffer exchanged the next day. Refer...Production of recombinant adeno-associated viral vectors for in vitro and in vivo use. Current protocols...tool for gene expression in mammals Use the PEI calculator for planning AAV packaging transfections Find...

...-3.13. To test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that...editing Hu et al. also engineered transcriptional activators using the xCas9 scaffold. With an NGG PAM, xCas9..., confirming the flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like...xCas9 3.7 fused to third generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing...a smaller increase in NGA editing. Adenine base editor xCas9 3.7-ABE also follows the same pattern, with...Plasmids for Mammalian Systems Learn about CRISPR History ...

...localization, dynamics, and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce...has gone into making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally ... protein fusions Transmembrane proteins, i.e. receptors and transporters, are integrated into lipid bilayers...spiderweb-like pattern of tubules can be grossly distorted into dense stacked membranes termed Organized ...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz... His interests include the quality control of secretory proteins in the Endoplasmic Reticulum and live...

...production is Komagataella phaffii (a.k.a  Pichia pastoris) because it secretes fewer of its own proteins...using a desalting column or an ultrafiltration concentrator. As the name suggests, you may also concentrate... to their parental hybridomas. Aggregation and storage Aggregation of antibodies may impact their quality...testing. An aliquot of our recombinant antibodies is stored at 37 °C for a period of two weeks. After this ...we are also cautious during the aliquoting and storage process to prevent dropped tubes. Now that your...Varley P, Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture...

... (NHEJ) and alternative end joining (alt-EJ). Inhibitors to essential components of both pathways have...events (Yang et al., Int J Mol Sci). Adding these inhibitors to your culture media prior to and after Cas9...introduction increases HDR, just make sure to remove the inhibitors within a day or two to not affect the viability... one step further, you can inhibit cell cycle regulatory proteins responsible for the transition out of...to fuse an HR protein to the nuclease. Early HR factors fused to Cas9 have been shown to increase HDR frequency...committing a break to performing HR before other factors were loaded onto the break, potentially shuttling...

...small-molecule inhibitors of a particular PPI. In such experiments, small-molecule inhibitors are tested ...because FRET depends on a number of hard-to-achieve factors. For example, FRET requires that fusion proteins...signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of the first...between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more difficult...library to screen PPIs among 450 transcription factors in live fruit flies. Although BiFC open-reading...

...available by both the Jacob Corn and Alex Schier laboratories. Delivering Cas9-gRNA ribonucleoproteins  An...This technique utilizes Cas9 proteins harboring receptor ligands (C in the figure above), which result ... potential complications, there are additional factors to take into account with these types of experiments...and purification, which will require additional laboratory reagents, equipment, and expertise. Final thoughts...production of the Cas9 protein and a gRNA in your laboratory, once these have been acquired, genomic editing...Central PMCID: PMC4289409. 3. Rouet, Romain, et al. "Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease ...

...Our story today starts with a bit of Addgene history... As you may be aware, Melina Fan, her brother ...get Addgene from a concept to an up and running repository. As they built the foundation for what Addgene...previous years, this event was held at Executive Director Joanne Kamen’s home- a testament to the familial... confident that their teammates and project collaborators have completed their work thoroughly. It’s kind...Veckerelli is the Administrative and Operations Coordinator at Addgene. In addition to her operational responsibilities...

...yourself needing to brainstorm with a team. One major challenge with these brainstorming sessions is avoiding...with unlimited time? Another strategy for your brainstorm session is to start with something completely... ideas that I have found helpful in my team brainstorming sessions.  Allow the group time to think about...This is why perfectionists tend to also be procrastinators.  If you fall into this category, get out of...our lab space, our protocols and even our Lab Inventory Management system. I hope these tips help you ...

...bacterial world produce an assortment toxins to claim territory or kill competing microorganisms, but did you ...as a counterselectable marker in the destination vector. Successful cloning occurs when the ccdB gene is...unmarked genetic modifications in the yeast Pichia pastoris. By combining both positive and counter selection...induced by methanol. Once linearized, the delivery vector can be integrated into the genome to introduce .../sok toxin-antitoxin system, they were able to monitor lacZ activity over 48 hours in vitro without antibiotic...marker for unmarked genetic modification of Pichia pastoris." FEMS yeast research 9.4 (2009): 600-609. PubMed...

...lysozyme to reduce basal expression levels; expression vector cannot have p15A origin of replication F- ompT...higher T7 lysozyme expression than pLysS; expression vector cannot have p15A origin of replication F- ompT...codons not normally used in E. coli. Expression vector cannot have p15A origin of replication F- ompT ...alleviates inclusion body formation. Expression vector cannot have p15A origin of replication fhuA2 ...Cis-repression of the E. coli T5 promoter (found on vectors such as pQE or similar), inducible under IPTG (...lac repressor on the pREP4 plasmid). Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15...

...signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner Rho GTPase, leading...to reveal even more insights. Super-duper lipid editor And if recruiting proteins to the cell membrane...phosphatidic acid signaling (such as Hippo, AMPK and mTOR pathways) and to introduce unnatural head groups...membrane or lysosomes and its activity can be monitored by phosphatidic acid probe GFP-PASS. B) Time series...Additional resources on the Addgene blog Viral Vectors 101: Optogenetics Additional resources on Addgene.org...Activity-based directed evolution of a membrane editor in mammalian cells. Nature Chemistry, 15(7), 7....

...know is your area code. Senators: To find contact information for your senators all you need to do is select...active research scientists (scientists who hold a doctorate degree, are currently working full-time, and have...edition. Cambridge university press, 1988. AAS: Historical Trends in Federal R&D NJ.com: Rush Holt: Lawmakers...Scott Pruitt says carbon dioxide is not 'primary contributor' to global warming The New York Times: With Trump...affected by Trump’s immigration ban Studies in History and Philosophy of Science Part: Democratic values...

...list from your current P.I., lab manager, or administrator containing all of the materials that you’ve ...financial values. All too often, we see early investigators making unnecessary short term sacrifices that...negotiations can still go horribly awry when several factors aren’t taken into consideration. 1. Always focus...solutions to reach your goals. For example, a junior investigator turned down a faculty position because none ...Happy Sciencing, Damien   Damien Wilpitz is a laboratory research management consultant and coach in Boston...research scientists create productive and manageable laboratory environments. His business skills enhance his...

...construct (about 8 in Brainbow-1.0 mice.) The combinatorial effect of multiple, independent recombination...woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene...Addgene also has two Brainbow adeno-associated viral vectors (AAV) available - AAV-EF1a-BbChT and AAV-EF1a-BbTagBY...of neural circuits, such as connections between motor neurons and the neuromuscular junction. In zebrafish...plasmids References: Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous...PubMed. Multiplex cell and lineage tracking with combinatorial labels. Loulier K, Barry R, Mahou P, Le Franc...

...combining AcrIIA4, a Streptococcus pyogenes Cas9 inhibitor, with the LOV2 photosensor from Avena sativa, ...-CRISPR plasmids Tagging human transcription factors with GFP Kevin White’s lab recently deposited a... of CRISPR plasmids to tag human transcription factors with GFP. As part of the Encyclopedia of DNA Elements...plasmids will help researchers map transcription factor localization and binding via ChiP-seq. Learn ...Learn more about ENCODE Find the transcription factor tagging plasmids If you have a new CRISPR tool you...

...Addgene with our Executive Director, Joanne Kamens. As a regular contributor of careers blog articles, ... our visitors made sure to tell us what we already knew about our Shipping Operations Coordinator: “Kory...teams at Addgene. An introduction to plasmids with Director of Biology Lianna Swanson was very popular, with...

... take into account chromatin context and other factors, they are easy to use and can reduce off-targets...nicking/cleaving of DNA occurs, not for deadCas activator/repressor experiments where only localization ...- GUIDE-Seq and integrase-defective lentiviral vectors - which rely on insertion of DNA by the NHEJ pathway...RNA-seq would be an appropriate method for an activator or repressor Cas. Similarly, Cas ChIP-Seq would... (e.g. in a gene in a similar pathway, a growth factor, etc.) this control is usually not very meaningful...and off-target risks will vary depending on many factors. Hopefully now you have all the resources you need...

...and luckily genetics is a sector which has been rapidly growing. These factors left me optimistic that ...and the pro’s and con’s of working in different sectors. Luckily, I have worked for a variety of different...options for going into parasitology but another major factor held me back; I do not have a second language. ...allowed me to try out working in many different sectors and to discover for myself what potential job opportunities... Are Open at Addgene Learn How to Choose a Good Mentor for Your Professional Development ...

...Chinmaya Sadangi, a postdoc at the University of Toronto. The Addictive Brain was founded in early 2018 ...Chinmaya Sadangi, a postdoc at the University of Toronto, created The Addictive Brain to spread awareness...non-scientists can directly approach scientists and doctors with their questions and comments.  Lastly, we ...Keep your message to the main three points of your story. Interested in learning more? Visit their website...Sadangi is currently a postdoc at the University of Toronto and founder of The Addictive Brain. Prior to this...

...signaling activity and cell phenotype. One method for monitoring live single-cell kinase activity is FRET, but... distance and the orientation of the donor and acceptor proteins, which can be difficult to predict. ...disadvantages when compared to FRET reporters. First, KTRs monitor global kinase activity that occurs in the cytoplasm...experiments. Third, KTRs are not well suited for monitoring kinase activity that occurs during nuclear envelope...the nucleus, there’s no nuclear localization to monitor). Whether you’re looking for a ready-to-use KTRs...

...here!   Marty LaFleur is a Postdoctoral Fellow in Arlene Sharpe’s laboratory at Harvard Medical School ...Cas9) hematopoietic stem cells with a lentiviral vector containing a gRNA. These transduced stem cells ...custom mouse strains and lentiviral expression vectors (Table 1). The four systems Name Function...G., & Sharpe, A. H. (2024). X-CHIME enables combinatorial, inducible, lineage-specific and sequential ...

...The first is the study of non-coding regulatory elements. Regulatory DNA function is typically inferred ...interested in exploring the function of non-coding regulatory elements in their native chromatin context. An...Bauer is a pediatric hematologist and principal investigator at Boston Children’s Hospital and Harvard Medical...PhD student at Harvard Medical School in the laboratories of Stuart Orkin and Daniel Bauer. He received...

... successful, internationally renowned plasmid repository that it is today. Although I wish I could take... attributed to the entire Addgene family; the depositors and requesting scientists, all of the Addgene..., Addgene supporters, and of course, Executive Director, Joanne Kamens, and founders, Melina Fan, Ken ...about her grandfather’s humble beginnings as an operator of a printing press. Just as her grandfather used...Joanne shared a moving thank you email from a requestor in Iraq (Addgene's first). After the speeches,...

...wanted users to be able to find plasmids and viral vector tools more easily. In the redesign, the new search...unique: community, sharing, and fun. The Addgene repository is built on the foundation of a community that...dictate the space. We wanted to comfortably tell our story, to get scientists excited about what Addgene has...redesigning the homepage, and striving to tell our story in better ways, we realized that Blugene needed ...you come to Addgene to find the plasmid or viral vector for your next experiment, that you can find what...

...  Dr. Sibby Anderson-Thompkins, Director of the Office of Postdoctoral Affairs at UNC, called her presentation...current postdocs will be academic principal investigators. She also pointed out promising new practices...increasing the value and priority put on good mentoring and applauded the new requirement for PIs to include...opened with a discussion of the Novartis industry postdoctoral program.  His strongest comments were about ...

... markers all tagged with mEmerald. Plasmids to monitor protein-protein interactions through multicolor... Baird said. “They are all designed in the same vector backbone with very similar restriction enzymes....Michael Davidson told the Tallahassee Democrat in a story about his life’s work and the stage IV lung cancer...shutter its doors. “We wanted to give all of these vectors to the scientific community and contribute in a...community," says Joanne Kamens, Addgene's Executive Director, "and to promote the great work of Michael Davidson...

...technologies, one might incorrectly relegate RNAi to history. The straightforwardness of RNAi is unmatched—unlike...cannot be used to investigate essential genes. Historically, gene silencing methods were thought to be afflicted...considerations, such as the starting material (DNA-based vector or purified RNA) and the initial state of the dsRNA... Although downstream work is required, several vectors can be found at Addgene that could serve as an ...an excellent start (#22988, #137884). Both vectors allow the creation of amiRNAs based upon miRNA precursors...method. Instead of using a fluorescent output to monitor RNAi, we engineered a positive selection reporter...

...chance vs. the factor that is being experimentally manipulated. Below are several core factors of statistics...variables – factors which are not affected by other variables (cause). Dependent variables – factors which...total crop production and the production window. Factorial ANOVA This type of ANOVA assesses multiple independent...sounds like a two-way ANOVA. And this time, it is! Factorial ANOVAs include two-way ANOVAs but can include ...

...making Laos the 112th country that we support. This story is a vivid example of how the global scientific ...advanced. Behind every publication is another great story, like researchers in India using Addgene-shared ...asked for a smaller size for our on-demand viral vector packaging service to better match experiment needs...Whether you're depositing plasmids, ordering viral vectors, or exploring our educational resources, you're...research. Thank you for letting us be part of your story, and for being such an important part of ours. We...

...memorable logo DNA was an obvious place to start brainstorming our new logo. It literally underpins our DNA-derived...sketched hundreds of ideas: Figure 3: Logo brainstorming. The winner shows a twist of DNA forming...so much more Addgene started out as a plasmid repository. Over the years, we recognized that what we offer...Addgene also offers other materials like viral vectors and antibodies. Plus, we’ve invested in data sharing...Addgene’s Blue Flame Award is prized among our depositors. Marking plasmids that have been requested over...