...libraries Learn about other members of the Addgene Advisory Board Additional Resources Learn a little...post-doc, Doench worked on using RNAi screens to learn more about cancer biology in Ed Harlow’s group at...and “with CRISPR, just the results are so much cleaner. The time frame from doing your primary screen ...Institute here Read other blog posts about CRISPR Learn more about the Broad Institute and the Platform ...

...rapid multi-part DNA assembly method. Read on to learn how to use it in your research! The starting point...The key is T4 DNA polymerase, which has 3′→5′ exonuclease activity. T4’s polymerase activity can be thwarted...available to polymerize, allowing the slower exonuclease activity to proceed. By including only one type...researcher can decide exactly how far the T4 exonuclease proceeds: at the first occurrence of the chosen...overhangs must not contain the dNTP present in the exonuclease reaction or polymerization will occur at that...

...illumination. This ability to reversibly bind and release a protein of interest in response to light allows...reaches the nucleus where gene expression changes lead to cell proliferation and differentiation. The lab...739201.  Additional resources on the Addgene blog Learn about using nanobodies to control protein activity...Read about RANbodies, reporter nanobody fusions Learn more about using the secondary nanobody toolbox ...Addgene.org Find nanobody expression plasmids here Learn more about optogenetics in Addgene's optogenetics...

...a team that created a novel form of AI that can learn new tasks and beat humans at the ancient Chinese... side you’re sure to have many opportunities to learn about and develop technologies that will have impacts...pulled tools from across the biological spectrum to learn more about the brain and neurological disease. I...better understand how aberrations in brain biology lead to altered function or maybe your favorite FRET-...pathways. Many scientists came to the Addgene booth to learn more about these tools and to tell us how useful...

...Addgene’s blog to learn about making your own competent cells and our protocols page to learn about bacterial...have studied how HGT occurs in nature and have learned how to introduce genetic materials into cells in... and can include plasmid DNA or fragmented DNA released into the environment. Various factors promote ...more viral particles. Eventually the cell lyses releasing the newly formed viral particles that can infect...

...the following information for a Cas enzyme: Nuclease properties (CasID): Cis/trans activity, PAM requirement... (one of the most frequently used Cas enzymes) cleaves both strands of its dsDNA target in cis and has...generates staggered DNA nicks at its target in cis and cleaves ssDNA in trans. In sum, CasPEDIA documents the ...along with its action on the substrate (nick, bind, cleave, mixed). This information represents the ‘big picture...Perhaps you are a structure junky who just wants to learn about Cas domains. CasPEDIA either directly has ...

...inspired you to perform this analysis. Having a clearly stated, specific goal for each analysis will help... currently working in) every time you use them, leave them in their own folder. Doing so will ensure that...same directory where it was run. This technique leaves a trail that further helps you keep track of your... entered is useful for beginners who are still learning the basics. However, these notes take up a lot...Read Our Tips to Keep Your Lab Notebook Organized Learn about Software You Can Use to Save Time and Track...

... a date or two for beer, coffee or meals with colleagues you know will attend the conference. Invite others...sure your name tag is always visible. Some people learn names by seeing them. It is OK to ask someone to...the same talk. Read more on this advice to “never leave an empty seat” here. If there are communal meals...especially if they are not in academia. Make sure to learn how to use LinkedIn properly. Then, follow LinkedIn... lunch twice turns a stranger into a friendly colleague. Recommended Reading & More: Scientist Networking...

...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab...a report to communicate your findings to your colleagues. So perhaps a better definition of “problem solving...challenges we encounter. As you spend time in a lab learning techniques and taking on independent projects,...opportunities to practice problem solving skills and learn to apply them flexibly in a range of contexts. As... organization Time management Managing a team Leadership Resources on Addgene.org Check out our careers...

...will never ever stop planning, perfecting, and learning, but there are a lot of things we actually don...much potential for service and contribution! Leadership On an organizational level, we keep passing...service and support. The Addgene management team leads with excellence. They are one of the main reasons...the Addgene blog Read our viral vector blog posts Learn more about Addgene Resources on the Addgene website...website Find plasmids for your research  Learn about our viral service Check out job openings at Addgene...

...conclusion of inter-homologue recombination was released. This blog has not been updated in response to...to understand what Shoukhrat Mitalipov and his colleagues have achieved, and how the field will move forward...correcting an autosomal dominant mutation in MYBPC3 that leads to hypertrophic cardiomyopathy. An adult male with... appealing for heterozygous mutations, it will clearly be much more challenging for homozygous mutations...editing rates vary widely from gRNA to gRNA, it’s unclear where this study would fall in terms of editing...

...possibly be made up of. The answer is… drum roll please… non-coding RNAs! You probably didn’t see that ...activation (CRISPRa) are powerful tools. CRISPRi uses a nuclease-dead Cas9 protein (dCas9) and a sgRNA to temporarily...targeting ncRNAs. For example, the Cas12a variant can cleave single-stranded RNA using gRNAs to specifically...Charpentier E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012...induced by CRISPR/Cas9 in the 5’ region of microRNA lead to its depletion and Drosha processing retardance...

...vector via Golden Gate cloning, perform an exonuclease cleanup, transform, plate, and pick. In short, the...found instead ones that were… pretty close, or at least close enough, you’ve found yourself with a common...Fragmid uses Bbsl, a Type II restriction enzyme that cleaves outside of its recognition sequence. This means...in duplicate, they were able to confirm that at least one plasmid was the correct sequence for every individual...

...Gag-Pol polyprotein, cleaves the proteolytic site connecting the two proteins, thus releasing Cas9. Nanoblades...Additional resources on the Addgene blog Learn about RNP delivery Learn about CRISPR delivery methods for mammalian...CRISPR guide Find CRISPR plasmids and resources Learn about viral production ...

...Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess...variants By Mike Lacy In 2022, Atsushi Miyawaki and colleagues identified a highly bright and stable fluorescent...just a few days later, Mohan Balasubramanian and colleagues showed the single mutation E138D is sufficient...is worth mentioning that Kiryl Piatkevich and colleagues generated yet another monomeric StayGold variant...-3188559/v1.  Neurogenetic alchemy: CAP-Mac unleashes the secrets of primate brains with brilliance ...

...ever like to write about a recent plasmid deposit please sign up here.  Here's what you'll find in this ...Caenorhabditis elegans on extrachromosomal arrays can lead to artifacts due to overexpression. CRISPR can insert...Ingaramo, Jerome Goudeau, Cynthia Kenyon, and colleagues at Calico Life Sciences. Like many split FPs,.... To resolve this issue, Shangqin Guo and her colleagues at Yale University fused a color-changing fluorescent...codon-optimized, engineered Streptococcus canis Cas9 nuclease that has a broad 5′-NNG-3′ PAM. Get the plasmids...

...scientific enterprise and the way it is funded and to learn tangible skills necessary for affecting change. ...eligible for overtime pay. For many postdocs this will lead to a raise in salary, particularly given that this... a rough road ahead for students interested in leading the process despite the recent advance. Similarly... treatment of trainee scientists as workers, at least for the purposes of labor laws, rather than as trainees...Addgene's Take Aways from the FoR 2015 Symposium Learn to Advocate with the Perfect Science Talk Find a...

...previous post from our Plasmids 101 series, we learned how the Cre-loxP recombination system can be used...DNA fragment using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment...reporter will be greatly reduced, allowing for clearer characterization of the location, morphology, and...containing FLEx technology. Read the genOway press release for more information. References: 1. Schnütgen ...Means of Genome Engineering Using FLEx AAV Vectors? Learn about Viral Vector Elements Read Our Primer on Optogenetics...

...Chromoproteins also enhance corals’ resistance to mass bleaching, the loss of their symbiotic algae, during heat...rhamnose-inducible promoter. The resolution step leads to the desired mutation and excision of the vector...mammalian cell expression here   Now that you’ve learned what chromoproteins are and how they can be used...blog Find our Fluorescent Protein 101 blog series Learn more about FRET Find protocols and tips for making...Blanquet RS, Phelan MA (1987) An unusual blue mesogleal protein from the mangrove jellyfish Cassiopea ...

...experience, I was still incredibly nervous about leaving academia. A lot of academics tend to view industry...postdoc experience, I’m going to share what I’ve learned and hopefully make your decision a little easier...publications, which may take a longer time to acquire. Learning about drug discovery: Unless your academic lab...market usually takes over a billion dollars and at least a decade of work. The number of steps, decisions...an academic postdoc, you’re often required to at least attempt to write grants. If you don’t enjoy or excel...

...hatching into their adult phase. It is important to clear out the parent crosses from the container before...sharing-oriented, and there are many resources to help you learn more about fly biology and fly-based experimental... and gene expression. In the next post, you’ll learn the fundamentals about how many of these resources...between pathogens and their hosts. It’s not always clear which side he roots for. You can try to convince...Methods for Site Specific Recombination in Drosophila Learn about Cre-Lox Resources on Addgene.org Find Drosophila...

...Identifying which genes are important for a phenotype can lead to a wealth of additional experiments investigating...consists of two components: the non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA...to enrichment or depletion of specific gRNAs can lead to skewed results. Selecting a cell type - Theoretically...validation practices, CRISPR libraries can help you learn a lot about your phenotype of interest. For more...Lentiviral CRISPR Libraries” (McDade et al., 2016). To learn a bit about how CRISPR/Cas9 can be used in other...

...dependencies  Take, for instance, a recent preprint released by the Boehm and Beroukhim labs at the Broad Institute...genetic dependencies of individual tumors, which can lead to new cancer targets and related biomarkers . Data...strongly correlated genetic dependencies that were clearly associated with European or East Asian ancestry... accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’...undercounted by approximately 20%. In some cases, this may lead researchers to deprioritize genes that could be ...

...our CRISPR Featured Topic Page Learn about RNA Targeting with Cas13a Learn about CRISPR DNA Base Editing...contain the RuvC and HNH domains responsible for DNA cleavage, so they cannot directly edit the genome. A Cas13...They hoped to find a stably folded ortholog that cleaved RNA robustly, unlike LwaCas13a, which must be stabilized... multiplexed manner to edit multiple RNAs. It’s clear that CRISPR RNA editors will open new doors for ...

...cells are lysed, RNAs and proteins are removed to leave you with DNA. This DNA is then washed, and either...purify DNA, cellulose also efficiently binds, or at least entraps nucleic acids. The Botella lab took advantage...around. Finally, purified total nucleic acids are released by dipping the cellulose directly into a PCR buffer...columns, suggesting that columns can be reused at least five times. DNA purification kits don’t come with...used plasmid purification columns could also be cleaned with 1M phosphoric acid and re-used, but trace ...

...and mascot Blugene (check back on December 5 to learn more about this!). I’ve assembled a series of screenshots...filter, this one from the ‘Requests’ category, will leave us with a single plasmid to consider. If you get...search. Simply click the ‘X’ next to any filter to clear it. Here’s a list of available filters to refine...great improvement for our website and we hope it leads you to exactly what you need with every use. If ...redesign. Additional resources on the Addgene blog Learn more about other features on Addgene's website ...

...your cells of interest. Cas9 RNPs are capable of cleaving genomic targets with similar efficiency as compared...levels shortly after transfection, and are quickly cleared from the cell via protein degradation pathways....known as nucleofection, which forms pores in the nuclear membrane, allowing for entry of a DNA template..... "Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing." Journal...the Addgene Blog Subscribe to CRISPR blog posts Learn about other CRISPR delivery techniques Find tips...

...contributed by guest blogger, Luke Lavis, a Group Leader at the Janelia Research Campus, Howard Hughes Medical... photoactivation, etc. These impressive advances lead to an obvious question: In this age of GFP and its...fluorophore can emit a limited number of photons before bleaching. The amount of information one can extract from...Luke D. Lavis. Luke D. Lavis is currently a Group Leader at the Janelia Research Campus, Howard Hughes Medical...PMC3246539. Additional Resources on the Addgene Blog Learn About Other Protein Tags Catch Up on Your Fluorescent...

...Resources on the Addgene Blog Learn to do Restriction Cloning Try SLIC cloning Learn about Gibson Assembly ... One key difference is the plasmid DNA must be released from the bacteria in order to serve as PCR template...reaction. You just need to lyse the bacteria to release the plasmid DNA by either briefly boiling the sample...Read about Choosing a Molecular Cloning Technique Learn to Perform a Bacterial Transformation Deposit Your...

...visualizing promoter activity, and much more. Read on to learn more about GFP, how scientists have evolved this...exist with only slight variations separating them.  Please note that many fluorescent proteins found on the...F64L; S65T, Y66W; N146I; M153T; V163A; A206K Cerulean F64L, S65T, Y66W, S72A, Y145A, H148D, N149I, M153T... Y145A and H148D Stabilizes the structure of Cerulean derivatives F99S, M153T, V163A Improves folding...visualize the effects of alterations to this activity. Learn more about optogenetics at OpenOptogenetics and ...