...efforts make a precise edit or insertion via a chemical modifier. We used these categories to see what... and the longevity of tools seen in Figure 3, indicates that the usefulness of popular CRISPR tools is...other countries, from Japan to Australia, are significant depositors and requestors. CRISPR tools are developed... into Addgene’s CRISPR data? Read our recent publication, “The Expanding Dissemination and Distribution...support for, this and previous Addgene CRISPR publications.  References and resources References Pyhtila...

...BLAST sequence databases from the dropdown menu. Typically, we use the default nucleotide database “Nucleotide...Organism box. This small piece of information can significantly reduce your wait time for blastn, blastp, and...” program for blastn so that you can extract practically every single reliable base pair from your results... the “Align two or more sequences” option to drastically reduce waiting time for search results. Optimizing...other labs speed up their plasmid and cloning verification steps and free up more time for using your plasmids...

...transforming a plasmid into the bacterium Agrobacterium tumeficans - a plant pathogenic species that stably integrates...Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes (Kyndt et al., 2015)! It sounds...selection markers (basta, hygromycin, kananamycin, tunicamycin) (Nakagawa et al., 2007). Alternatively, if you...plants: Lots of plants produce lots of seeds. I typically use 8-10 plants per construct I transform. More...National Academy of Sciences of the United States of America. National Academy of Sciences, 112(18), pp. 5844...

... of these techniques have in common? They all typically require secondary antibodies, frequently of the...in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug...antibody-containing hybridoma supernatant is only $40, significantly cheaper than commercially available antibodies...

...addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.     Figure...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties... a concentration step prior to being frozen. Specifically, lentiviral particles are collected from the... Addgene's Data Hub Digital Droplet for AAV Quantification Viral Vectors 101: An Introduction to AAV References...

...the Society for Advancement of Chicanos/Hispanics and Native Americans in Science’s (SACNAS) annual conference... written up in mSphere. A post-meeting survey indicated that the program was largely a success, but also... even more valuable program to Microbe 2020 in Chicago.  Finding our place in networking and mentoring... career scientists, students, and those from historically underrepresented groups. Through such mentoring...

...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...

... serotype, are particularly challenging. Their efficacy in any given system can be affected by any number... the Addgene repository can save researchers significant time and removes a variable from the lengthy ...available in the repository. GCaMP, a calcium indicator (biosensor, by Addgene’s categorization) was first...trends in our own repository data, giving us significant insight into which vectors are likely to be of...Waxmonsky, N. C., Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR...

...a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such as horseradish...include undesirable molecules that may bind non-specifically to assay components and increase background ...staining. Direct ELISAs also lack the signal amplification afforded by secondary antibodies and thus are...antibodies are used.  Competitive ELISAs Scientists typically use a competitive or inhibition ELISA when measuring...While the precise setup of the method can vary, typically users first coat the multiwell plate with a reference...

...gives Snapgene great exposure to the community of biological scientists who use Addgene everyday. To get this...hesitation to ‘approach strangers’ and learn how to communicate outside my scientific expertise. Organizing a...will lead! Read our blog post about fostering communication in remote collaborations! Stay in touch: Maintaining...connection is to make sure you keep open lines of communication with colleagues who move on to new organizations...Universities themselves can help you do this through practical tools like alumni networks. I find it useful to...

...post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  The advent... than ever to make precise, targeted genome modifications. Cas9 has been modified to enable researchers...uses a “nicking” Cas9, in which one of Cas9’s two critical enzymatic residues has been converted to an alanine...instead.  Choosing a Cas9 for site-specific modifications Site-specific deletions and knockouts Some experiments...Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic a patient mutation...

...Why pseudotype? Lentiviruses and retroviruses typically target immune cells with their wild-type envelopes...out this review article published in Reviews in Medical Virology (Li et a., 2018).   Figure 2:...The glycoprotein will be incorporated into the replicating rabies virus envelope, travel retrogradely, and... Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene...Restriction of Transsynaptic Tracing from Single, Genetically Targeted Neurons. Neuron 53:639–647 . https:/...

...even when larger vector genomes are packaged, the physical size of the genomes is still ~4.7kb. But despite...overlapping strategy has a region of homology, typically ~400-1400 bp long between part A and B of the ...overlapping split vector had an improvement in muscle physiological performance.  2. Trans-splicing Overview: For...and may need to be optimized for each unique application (Trapani et al., 2015).  Expression of smaller..., was 81% of that from a single vector, but significantly greater than the expression from an overlapping...

...many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous...combining these two strategies allows you to specifically incorporate homo- or heterozygous mutations....in the field at the time but had never been systematically analyzed: introducing silent CRISPR/Cas9 blocking...efficiently incorporated during HDR. We decided to systematically characterize this phenomenon and found a general...University. He currently works to understand pathological mechanisms of Alzheimer’s disease by developing...

...favorite twitter #agarart accounts, Petri dish Picasso (@PetriPicasso) came together and delivered very impressive...are some of our 2017 contest winners:   Chicagoland's Community Biology Lab (@ChiTownBio) and one ...Mackin Lab from University of Colorado -Anschutz Medical Campus (@CSD_CUAMC) caught a case of holiday cheer...

...simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent ...on both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best with insertions of 2 kb...models varies from 8.5-100% of pups born, with typical efficiencies of 30-60%. Quadros et al. attribute...2.5x more animals than i-GONAD and is a more technically difficult process. Overall, the main drawback... out our CRISPR Guide Find Plasmids for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression...

...any molecular biologist Tips for protein purification Replica plating Anti-CRISPR Retroviral vectors Career...source of information on recent advances in the biological sciences and techniques that simplify and expedite... help hone your blog post for our audience of biological scientists from a variety of different fields...

...Investigator III at Novartis, a multinational pharmaceutical company based in Switzerland. After completing...completing her PhD in Chemistry and Chemical Engineering with Harry Gray at Caltech, Crystal moved on to a ...Chapter 7: How Crystal developed her science communication skills and how they've helped her at Novartis...scientific teams at the Novartis Institutes for BioMedical Research to develop novel biologics for the treatment...

...Addgene podcast, we sit down with Alex Chavez, a Clinical Fellow in Pathology at Masachusetts General Hospital...Church and Collins labs at the Wyss institute for Biologically Inspired Engineering. Alex is the first MD PhD...and explains why he loved the first 2 years of medical school. 7:05 - 10:55 : Alex's focus during graduate...postdoc 12:01 - 13:21 : The Wyss Institute for Biologically Inspired Engineering and why Alex decided to...

...acquires through Addgene to produce molecular biological tools like Taq polymerase, T7 polymerase, EcoRI...scientists to continue to do excellent work in the biological sciences. Future awardees Now that this first...concluded, Addgene and Seeding Labs will be opening applications for a second round of plasmid awards on June...excited to discover what new and creative ideas applicants will propose for plasmids from the Addgene community...

...infectious disease once limited to small pockets in Africa has spread to urban areas and across the ocean....percent of the most recent cases, primarily in West African countries. Even that grim estimate might prove ...protein present on the virion surface – making it critical for viral attachment and fusion. Once inside a...glycoprotein’s role in infection also makes it a critical component of vaccines and the target of neutralizing...

...the user. This approach is useful for several applications as will be discussed below. The software could...has a maximum of one gene knocked out. In this genetically heterogeneous population, cells behaving differently... you could majorly accelerate progression into clinical trials. Secondary screens  If you have generated...genes you’d like to test for phenotypic effects. Typically the phenotype would be studied in vitro, but there...contain multiple gRNAs targeting all genes in the indicated species and were suggested to be the best gRNAs...

...analyzing your cells using flow cytometry, you are typically measuring the presence or absence of certain markers...reporter, such as GFP or mCherry, through genetic modification. As your protein of interest will only occur...free amines on and inside the cells and are thus typically used for distinguishing live from dead cells. ...describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab. Let's say I am culturing...Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative...

...Antibody characterization is critical to enhance reproducibility in biomedical research. eLife, 13, e100211...tell only part of the story. This year, we made significant strides in making science more accessible and...launched AAV Packaged on Request, a service that dramatically expands and simplifies your viral vector options...

...promoter. It can be used to drive Cre expression specifically in neurons. AAV Delivered Cre - A useful tool...Indeed, this AAV provides you with a means to specifically activate expression of any Cre-dependent gene...pmSyn1-EBFP-Cre Of course there are also more complicated ways to use this particular AAV. For instance...Additionally, you could use the retrograde form to specifically express Cre in neurons with axons that extend...

...service. A small subset of AAV plasmids will have technical or legal restrictions that make them ineligible...be reviewed by Addgene for legal approvals and technical compatibility with our production process. This...request is approved, you will receive an email notification that your request is ready to order. An “Add...found in our in-stock preps. If a request has technical considerations that may affect expected titers...

...incredibly useful molecular biology tool, but like any biological system, it has certain drawbacks. First, the ...Gerald Rubin’s lab sought to make complex genome modifications in Drosophila using multiple recombinases. To...offspring carried both the recombinase and GAL4, indicating that the recombinases were inherited normally...Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of the mouse brain. Rubin...

...  “This enables you to do customized genetic modification on a scale that was really not possible before...genome engineering with CRISPRs was limited to modification of a few loci at a time,” Wang said. “It was... lentiviral libraries and their demonstrated application offer proof-of-principle that CRISPR-Cas9 can...aimed for 300 cells per construct, but in some biological systems one “might need larger numbers.” Further...

...: Find sequences for plasmids assembled from publications and commercially available sources, as well ... as expression patterns, post-translational modifications and polymorphisms Codon optimization tool: To...making it ready to copy and paste into other applications Primer design tools Many of the above-listed... well as primer design for different cloning applications. Primer3 and Primer3Plus: For general primer...

...time for me to complete an online social media certification on social media data, and I was given a fantastic... occasional TikTok. I was also responsible for typical entry level tasks such as stuffing new depositor...connections with coworkers seem, in retrospect, nonsensical. I have found the people at Addgene have become... at Northeastern and Addgene co-op studying communications, marketing, and global fashion studies. She...

...symposia on Sunday afternoon were dedicated to science communication through art. Addgene Advisory Board...page and, just a few weeks ago, we attended the American Society for Microbiology's annual meeting (ASM... Jessica Welch...

...service organization like the Red Cross or the American Cancer Society, or maybe you think about a local...services we provide like quality control, curation, technical service and open-access educational resources....decisions at Addgene If you’re ever curious what specifically drives the decision making process at Addgene...Gizela, Henry, Ina, Inna, Ishrat, Jane, Jason, Jessica, Joel, Jonathan, Justine, Karen, Kate, Kayla, Kelly...

... perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal...How else can antibiotics be used in the lab? Historically, antibiotics have also been used to disrupt ...cloning. In these instances, the cloning vector typically has two separate resistance cassettes and your...and can be used in place of Ampicillin in most applications. Antibiotics vary in their sensitivitly to heat...

...preparations have worked well in my lab, including in applications where we have delivered them in vivo to the ...Roth’s lab are already available allowing you to chemically control neuronal activity while studying your...distributing new types of materials. At the time of publication we’ve already fulfilled more than 140 orders ... service as we continue to develop it. More communication and sharing will only help research advance....

... lab at ETH Zürich. “Instead of a graphical abstract, it’s a musical abstract,” says Di Ventura, a professor...in @nchembio Title: Bye bye L-arabinose drive. Dedicated to Edo, Armin and @KhammashLab. I hope you all...reached the level of expression needed for most applications.   Figure 1: With blue light, the VVD...

...physiology. Ribosome profiling Ribosome profiling, a biochemical method developed in 2009, takes a “snapshot” ...translation While ribosome profiling co-opts the physical location of the ribosome, TRICK (Translating RNA...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...studying translation in various cell types and physiological states. References 1. Ingolia, Nicholas T.,...

...represent the fraction of that total with the indicated function. One huge reason CRISPR has become such...Chavez, researcher at the Wyss Institute for Biologically Inspired Engineering and member of the Addgene...achieving the gRNA representation recommended in the publications associated with these libraries. Please note...particular, the Church lab at the Wyss Institute for Biologically Inspired Engineering and the Zhang and Doench...

...microorganisms based on metagenomic data. MerMAIDs (Metagenomically discovered Marine, Anion-conducting and Intensely...publicly available metagenomic datasets with biophysical studies and the potential of mining datasets ...plasmids at Addgene Opperman J et al., Nature Communications, 2019. PubMed PMID: 31346176. Using LADL to...CIBN), which they call the LADL Anchor. Using specifically designed gRNAs, they can then recruit these ...

...ll need a few controls specific to flow as an application. These controls will allow you to distinguish...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...commercial antibody pages. Flow cytometry can be a complicated and daunting process. Controls like single color...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines already expressing Cas9...