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... Protocol Addgene's Video Antibody Purification Protocol Addgene's Antibody Guide References Trimmer JS...the periplasmic space to be oxidized and form disulfide bonds (Frenzel et. al., 2013). If you’re using...stained, the heavy and light chains become visible alongside your ladder and a protein standard. The density...calculated using imaging software. Any bands on the gel beside the heavy and light chains can also be measured...multiple contaminating bands or a few strong ones, consider increasing the number of washes performed during...

...validated for BiFC (Kodama and Hu, 2012). Having a rough idea of how your PIPs might interact will allow you to...each PIP and empirically test which combination provides the best fluorescent signal. As for linker sequences...experiments. In either case, overexpression should be avoided because it can generate high background fluorescence...critical to the success of BiFC experiments. The ideal control is a mutated variant of one or both of your...that you empirically test a number of mutants to identify those that inhibit BiFC in your cellular system...endogenous PIPs is expressed at increasing levels alongside your BiFC constructs. If your PIPs actually interact...

...Vector Plasmids Read Our Lentiviral Vector Guide Read Our AAV Guide  ...the cell when the appropriate viral proteins are provided in trans (see Other Important Components of EBOV...separate plasmids and are co-transfected into cells alongside a plasmid encoding the minigenome. These genes...The power of reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen,...Posts on Viral Vectors Check out Our Scientist's Guide to Ebola Contribute to the Addgene Blog Resources...

...mission by hard work, we genuinely enjoy each other’s ideas and company. The Addgene summer picnic This past...though she generously offered. Instead we picniced outside of our new office space on the nicely manicured...together is that when it’s time for fun, everyone is confident that their teammates and project collaborators...events over the years. To keep the organizational side of things from taking too much of your time, scroll...(or more popularly- cookies). 3. Do something outside of your ordinary environment Team building events...something fun happening! Addgene actually has chimes outside of our break room. A ring of the chimes means something...

...substances are part of toxin-antitoxin systems that are widely present in bacteria. They consist of a toxin which...systems have been used as biological tools for a wide range of applications in molecular biology or in...Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is GatewayTM cloning, ...toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many protein-based toxin-antitoxin... endonucleases cleave DNA at or near specific nucleotide sequences. To counter this, methyltransferases...methyltransferases add methyl groups to bases at specific nucleotide sequences, preventing the cognate restriction enzyme...

... DNA sequence to encode your peptide of interest and produce that peptide in your organism of choice. ...as easy as it sounds. Even for relatively short peptides, there can be many possible ways to encode them... the native organism.” This is possibly a better idea and has been used successfully in the past (Angov...2008), but there are still many more features to consider when designing a full gene. A non-exhaustive list...PubMed Central PMCID: PMC4794256. This review provides a great overview of codon usage bias 8. Tuller...

...have accumulated more than 300,000 individual nucleotide sequences. More than half of these sequences ...assembly algorithms meant that we already had a clear idea of the necessary steps to perform:      ...entire package to run. The small, portable system provided by a container is sequestered from other programs...container because of the reproducibility containers provide and for convenient compatibility with our internal...complete pipeline however, required more than just identifying criteria that we needed. Prior to using Nextflow...that the desired data was available so we felt confident we were using the input and output to link the...

...with no additional off-target edits, making them ideal for size-limited AAV delivery. PE6c and PE6d are...modalities and has the potential to reduce negative side effects. This platform offers a promising approach... TransitID By Mike Lacy Proximity labeling can identify protein interactions or map a local proteome, ...dual-enrichment and mass spectrometry workflow identifies proteins bearing both labels (which had therefore...locations. C) Enrichment of dual-labeled proteins identifies proteins that had been trafficked from the “source...bioRxiv, under CC-BY-NC 4.0 license. This system provides a unique new way to record and quantify the dynamics...

...Fluorescent and bioluminescent reporter genes are widely used to study gene expression in cells. But these... by tissue. Ultrasound, on the other hand, is a widely available and inexpensive imaging modality with...high spatial and temporal resolution, making it ideal for visualizing gene expression in vivo.  But what... be easily expressed heterologously by bacteria inside the body. So we screened gas vesicles gene clusters...origin of replication (p15A) worked best under a wide range of inducer concentrations (0.1-1% L-arabinose...bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the tumors was overlaid with the anatomical B-mode...

...600 Cas9 orthologs and found that they could be divided into two groups: longer orthologs approximately...Split inteins are a pair of naturally occuring polypeptides that, when at the ends of two proteins, mediates... CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566:218–223 . https://doi.org...2014) Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell 156:935–949. https://doi.org...doi.org/10.1038/s41467-018-08224-4  Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur... Resources on Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...might not localize to the right places, which can provide misleading information on where exactly a structure... via BirA and AviTAG. pMAK contains the signal peptide of MBP, followed by the desired nanobody (NB) sequence...sequence, an AviTAG, and 6xHis tag. The MBP signal peptide allows the NB to be exported to the periplasm of... the expression of nearly any gene via specific guide RNA (gRNA) sequences. But these approaches rely ... viral aliquots from our plasmid collection to provide ready-to-use viral preps. Here are some of the ...

...others can provide you with the highest form of flattery - imitation! Use the MIQE guidelines (Bustin, ...primer and be at least 25 bp away from each primer, ideally located close to either the forward or the reverse...across the plate is fine; a 5% positive error on one side and a 5% negative error on the other will introduce...triplicates in multiple configurations can help you identify pipetting or evaporation errors. And if time allows...look at their 551 page e-book on applying MIQE guidelines! With these kinds of resources, you will soon...Shipley GL; Vandesompele J; Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative...

...and find our guides, plasmid kits and other resources for microbial research. Kits, guides, plasmids, ...biology, gene cloning and expression, as well as guide pages for Synthetic Biology and Bacterial Expression... our Genome Engineering Overview and our CRISPR guide to find out how to use genome editing techniques...Expression and Insert Species filters on the right-hand side of the search page to narrow your keyword search...

...Twitter for the official launch!  Videos Look out for our next protocol video, “Western blots,” coming soon...also be building our video library all summer long, with additions to our other video series.   And if you...as excited as we are! Come join us in our blog, video, and social media endeavors - in between some much-deserved...

...frequency three orders of magnitude higher than identical targeting vectors delivered through transfection...AAV effect remains unclear, it is likely not a coincidence that the AAV genome is composed of single stranded... selection, individual gene edited clones are identified by PCR. Then, if necessary for subsequent experiments...disadvantages to the AAV-based approach that must be considered when deciding whether to use AAV or CRISPR for...for the generation of knockins, it is not always ideal. Because of these limitations, the AAV approach ...variety of cell types, now you know that AAVs can provide a powerful boost to genome editing experiments....

...for DMD treatment. Clinical trials have used oligonucleotide exon skipping (OEN) to remove mutated exons... dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and...Dystrophin and genome editing Since complex oligonucleotide treatment comes with many challenges, researchers...rigorously examined over a long period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should be ...Covello G, Denti MA. Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases... Resources on Addgene.org Check out Our CRISPR Guide Pages Find CRISPR Plasmids for Your Research Find...

...toolkit New Cas protein identified Jennifer Doudna’s lab recently identified Cas14, a family of small...or DNA. Now they have developed this further to provide multiplexed detection, higher sensitivity, and ...of DNA Elements (ENCODE), a consortium aimed to identify functional elements in the human genome, these...sheet Resources on Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited...

...scientists outside of the US now use the repository – about 50% of plasmids are shipped to labs outside of the...plasmid shipping look like at Addgene? Here's an inside peak into the daily workings in Addgene's lab. ...world... Where did 500,000 plasmids go? Far and wide – that's where! Check out the map below which depicts... improve the repository. We love being able to provide a useful (and growing) resource for our favorite...

...Plasmids 101 videos and needed a break so they're hanging out with Aavery in this Zoom video background....background. Want the purple bacteria video background with mascots Blugene and Aavery? Download the 720p version...our virus backgrounds. Addgene’s viral service provides ready-to-use AAV and lentiviral preps! Download...DNA background. Or get the same background as a video background! (720 px or 1080 px).         Do you...

...library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If you need help cloning...v2, you can take a look at the protocol kindly provided by the Zhang lab. Even though lentiCRISPR v2 has...of matrix metalloproteinase-9 secretion, and identifying lysine methyltransferase substrates. We’re incredibly...Shalem, and Feng Zhang. "Improved vectors and genome-wide libraries for CRISPR screening." Nature methods ...Resources on the Addgene Website Visit our Lentiviral Guide Pages Find Lentiviral Plasmids Find CRISPR Pooled...

...Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could help, or harm, your experimental...tools, where modification of cellular activity outside of your experiment site or circuit can occur. Promoters... promoters  This type of promoter allows you to decide when to turn on, or turn off, gene expression by...promoters to begin testing, you’ll next need to decide how much virus to put into your system, which is...injection volume.  There are a few factors to consider. First, the titer required is inversely related... posts in our Viral Vectors 101 series can help guide you with these, so keep reading! Good luck, and...

...when enhancers were active or silent, they could identify the target genes of previously uncharacterized...has made targeting much easier! With the help of guide RNAs, fusions between Cas proteins and the epigenetic...this fusion protein allows for LSD1 targeting to a wider set of genomic targets. These two vectors are part...CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair... the Addgene Blog Cas9 Activators: A Practical Guide CRISPR Between the Genes: How to Experiment with... Resources on Addgene.org Check out Our CRISPR Guide Pages Find Validated gRNAs to Target Your Genomic...

... coming out of MITs Human Dynamics Laboratory identified five characteristics of very successful teams...this series). Members carry on back-channel or side conversations within the team. We are not talking...contribute. Members periodically break, go exploring outside the team, and bring information back. No team ...colleague: MBWA "Manage By Walking Around" – don't hide in your office. Stop by instead of emailing.    ...volunteer constructive feedback and suggestions. Consider offering rewards (gift card, afternoon off, etc...

... a very different schedule. Fortunately, I was provided with a detailed training schedule that helped ...it relates to other teams at the company. This provides new (and old) employees with information on how...possible to work the remainder of the three hours (outside of the core hours) at a time that works for you...transition into a traditional work environment was aided by the efforts being put in by Addgene to ensure...blog.      More resources on the Addgene blog A Guide to Getting Started in Undergrad Research Tips for...

...mostly because of this article: “A systematic identification and analysis of scientists on Twitter.” This...Scientific Disciplines on Twitter. Twitter users were identified as scientists if they had a scientific title ...scientists within their fields than scientists outside their fields Figure 6: Follower Network of Scientists...follow scientists within their field rather than outside their field. Lower: Follower networks of subgroups... Y., & Sugimoto, C. R. (2017). A systematic identification and analysis of scientists on Twitter. Plos...

...requesting and ordering. This split allows us to provide researchers with increased choice and flexibility... Plasmids available for Packaged on Request consideration will have a banner on their material page. Interested...testing, and viral genomic sequencing to confirm the identity of the packaged DNA and serotype. Please note ... in-stock preps. If a request has technical considerations that may affect expected titers, a customer...1–2 days (USA); 3–4 days (international; varies widely by country and/or distributor.)  You should plan...

...certainly hope not! One of our core values here is to provide unmatched customer support, as this is vital to...exact problem, and it’s called “self-service.” The idea here is to build features that allow users to help...general question or concern about the answers we provide, within the widget you also have the option to ... we’ll be continually improving the support we provide based on your valuable feedback. This is Addgene...first. Resources on the Addgene Blog Read other Inside Addgene Posts Learn about New Features on Our Plasmid...

...joined by Addgene Scientist Matt Ferenc (one of our resident CRISPR experts) at the 2014 Johnson & Johnson’...up to the discovery of the mechanism of dual-RNA-guided DNA cleavage in adaptive bacterial immunity and...Dounda and Charpentier met at a 2011 conference and decided to collaborate on what was a “curiosity” driven...thanked Martin Jinek and Krzysztof Chylinski (who coincidentally speak the same Polish sub-dialect making their... Technology? Learn More: Addgene's CRISPR/Cas9 Guide History of CRISPR Cas - A tale of survival and evolution...

...post-grad plans, but in the end, writing won out. “I considered teaching at a liberal arts school but I’ve always...Iowa, but kept up a freelance writing hustle on the side. “I actually stumbled upon, about 6 months in, this... was for Cactus Communications, a company that provides scientific editing services. “I was working during...At the end of her postdoc, Caitlin had a clearer idea of her career goals: “My plan was to go into editing...landing her job. All of the hard work she’d put in outside the lab had paid off. “The combination of the editing...

...biosensors When choosing a biosensor, you first need to decide the best way to track or measure the process you... of small molecules like arabinose or hydrogen peroxide or ions like calcium and zinc. Some biosensors...compatible with your lab and experimental setup? Consider what kind of instrumentation you have available...components of individual cells Separation/identification of fluorescently-labeled components Fluorescence-activated...Optogenetics Plasmids Visit the Fluorescent Protein Guide Pages ...

... mapping where a protein binds genome-wide, and has been widely applied in many model organisms and cell... Enter CUT&RUN, an enzyme-based protocol for identifying DNA binding patterns that overcomes some of the... degree of signal to noise is desired. It also provides an excellent additional measure of DNA binding...technique will quickly become a standard method alongside ChIP. Many thanks to our guest blogger, Matthew...

...clinical data about a disease or therapeutic Side-by-side comparison of the same experiment from different...makes your insights discoverable. Watch this quick video to learn how ReFigure works and continue reading...Apps to Save Time and Track Your Experiments Use video to share your science at We Share Science Learn ...with Addgene Find Molecular Biology Protocols and Videos Find Other Molecular Biology Resources ...

...Visit our viral vectors guide Find our chemogenetics and optogenetics science guides ...Chemogenetic approaches excel at broad tissue or system-wide targeting, as the ligand can reach target cells ...optogenetics. If broad control of whole circuits or tissue-wide systems is necessary, use chemogenetics. Note that...approaches suffer from unique sets of off-target side-effects that should be carefully controlled for ...

...service that we, as scientists, provide for journal editors to help decide whether work is suitable for ...of the 18th century. Yet the peer-review was not widely adopted by the scientific community until the mid...peer-reviewing journal submissions since 2015 for a wide variety of journals. Since then, I developed a routine...submitted paper in addition to the journal-specific guidelines.  Peer-review step 1: The first read At the first...

..., Addgene will have unique Research Resource Identifiers (RRIDs) for each antibody, making it easy for...topics are discussed on Addgene’s blog and Antibody Guide, which include information about immunofluorescence...suggestions about how to make the resource better or ideas for new materials to add to the collection, please...find useful antibodies for your research. Please consider sharing your experiences with antibodies from ...Introduction to Immunofluorescence Addgene’s Antibody Guide The project is supported by a NIH BRAIN Initiative...

...transformation fall into two main categories: antibiotic/herbicide resistance and fluorescent proteins. Selection...undifferentiated plant cell culture), which is great for a wide variety of applications. But some techniques require...CRISPR-Cas9 gene editing, transformation may be identified by resistance to an antibiotic. Successful editing...therefore, are useful for research, and they are identified by a phenotype caused by the segregating mutation...these mutant plants. Using RUBY to simplify the identification of these mutants will speed up my work and ...

...developed and tested by ABRC staff. These kits provide in-depth, ready-to-teach lab protocols and supporting...supporting materials such as instructional videos and datasheets. The remaining kits were donated to ABRC...cycle, and is relatively easy to grow, it is an ideal organism for teachers looking to expand their science...outlined in ABRC’s kits vary by module. As a general guideline, experiments that include growing plants require...available via e-mail and phone to answer questions and provide support. Growing capacity - ABRC outreach today...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...a new CRISPR screening method that makes use of guide RNAs with internal barcodes (iBARs) in their loop...experimental replicates for each of the gRNAs. The idea was to assign four different barcodes to each of...covers every annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for... and can serve as a drug-screening platform to identify new antifungals that target calcineurin. Current...

...demonstrate that 85% of leaving postdocs pursue a career outside the traditional academic silo to tenured professor....  Here are some questions and perspectives to consider. What do academic and non-academic careers in ... a greater sense of autonomy - there are fewer outside influences on your topics of research or course...move around every 3-6 years—a short and exciting ride Complicated matrix structures are common Small... a scientist you enjoy the most. Having a better idea of where you want to go will help you focus on the...

...useful, chemical fluorophores can still be used in a wide variety of experiments. Read more to find chemical...potential applications of R-bodies and to find a video of them in action. AAV: A Versatile Viral Tool for...interest to a variety of cell types in-vivo. These are widely used by the optogenetics and chemogenetics communities...pathways using light and chemicals respectively. Didem Goz Aytruk from Connie Cepko’s Lab at Harvard Medical...AAVs function and indicates important things to consider if you’re getting started with them. CRISPR Pairing...