... months have in store for us. If you’re thinking about making the switch from academia to entrepreneurship...entrepreneurs. To make things more interesting, we are working in a new technical discipline that nobody understands...few jobs available. So, we spent a lot of time talking about what we could do to make ourselves marketable...marketable to the wider world and occasionally thinking about how we could bypass the establishment hiring...

...broken down into different types, with Cas3 systems making up type I, the most abundant type in the Class ...consistent expression can still be a challenge. A multitasking Cas Most Cas effectors have one job — Cas see...away at both the non-target and target strands by making cuts every so often. Cutting both strands ensures...carried out much like the name implies — after nicking, Cas3 reels in the non-target strand of DNA much... for Cas3’s biggest trick. Cas3 likes to bluff, making you into think it’s more powerful than it is. The...

...a bench scientist would looking at a genome by eye. We came up with a ranking formula that does just this...In the rest of this article, we'll explain the thinking behind the tool, its key features, how it works...information from Crispresso plus a summary per batch. Ranking guides for HDR in-depth In other tools, guides ... engineer with the Chan Zuckerberg Initiative, working on assignment at the Biohub in San Francisco. Before...

...You’ve spent days and weeks thinking of an amazing project. You’ve written your protocols, designed your...enzymes laying around the lab. If not, you’ll be looking at about $50-$60 per enzyme. Once you’ve got a ... of the restriction enzymes and overall you’re looking at a price of a few dollars per reaction for traditional...relatively new to the cloning game, however, or simply looking for a more streamlined approach, you might want...inserts: There are some cloning methods that make working with multiple inserts a breeze. Among them are ...

... they become confluent, taking both early- and late-arising clones, and making sure we always maintain...densities - adding to the expense of dilution cloning. Taking a page from bacterial cloning, we reasoned that...cloning rings. A picture of a 60 mm dish ready for picking is shown in figure 1.   Find Additional Useful ...firmer 2% agarose gel solution discussed above for picking. Many thanks to our guest blogger Iris Lindberg...

...Additional opportunities: Tracking intracellular pathogens In addition to tracking the location of proteins...University of Queensland) to observe membrane trafficking events in vivo. Locate your protein and see who...your images using subjective judgement by simply “looking at them”. Modern cameras and image analysis software...limited by the wavelength of light and, practically speaking, for common lab microscopes the detection limit...

...alleles. Although CRISPR is very good at making knockouts, knocking in large sections of DNA can be more ...negative selection marker like the HSV thymidine kinase (HSV-tk) is included just outside one of the homology...” the resistance gene through the insertion of flanking LoxP sequences, you can remove the gene through...described in the preceding sections are simple ways of knocking out a non-essential gene of interest. However,...

...Despite these grim statistics, new strategies for attacking cancer are yielding promising results in the laboratory...universally-dividing behavior of cancer cells, attacking the cellular machinery necessary for division....in the field of cancer research has focused on picking apart the complex cellular pathways involved in...found across all TCGA tumor samples is represented, making the results of any such study as clinically relevant...Addgene's cancer collection pages Scientists are making progress. New resources and technologies become...

...co-founder of the BioBricks Foundation, an organization working to advance biological engineering openly so as ...thing at a time. What the BioBricks Foundation is seeking to accomplish is to make the engineering of biology...is in reference to George Boole who in 1854 was thinking about logic and analyzing human language and observed... be made freely available? Endy: When we start thinking about languages, humans have two types of languages...really plugging into the "stuff sector" if you will, making chemicals and materials, and we anticipate plugging...

...using the application correctly is imperative. Picking your chemistry  The two most common qPCR chemistries...thermodynamic and experimental parameters (neighbor stacking energies, loop entropy effects, cation concentrations...type (such as here, here, or here).  If you’re working with samples that have not been documented in the... plateau stage after exponential amplification, making it difficult to properly analyze your data.  To...experiment! The model assumes the amplification kinetics of the target and control genes are fairly equal... e-book on applying MIQE guidelines! With these kinds of resources, you will soon become a qPCR master...

...Science: What the Future Holds and the Scientists Making It Happen, is an exercise in restraint. The very... revelations. The issues he discusses have been making the rounds in journal editorials, science social...likely range from mildly surprising to genuinely shocking. Mainstream media outlets have addressed CRISPR...recontextualized some areas of science that I’ve been thinking about a long time. For the scientists who like... Eric J. Perkins...

...Working with AAV vectors in the brain and peripheral nervous system presents a special challenge to scientists...answered this question and used it to publish!  Working with AAV vectors in neuroscience Let’s consider...consider my first challenge when I started working with AAV vectors. I wanted to use an AAV vector to express...label neurons transduced by each serotype. Working at Addgene has shown me this was far from an isolated...Results from a survey conducted by Addgene in 2021 asking scientists what data would improve our AAV Data...

...PMCID: PMC110254. 3. In-vivo RGB marking and multicolour single-cell tracking in the adult brain. Gomez-Nicola...pre-clinical/early clinical stages. Researchers are also working to make lentiviral vectors safer. Since lentiviral...Winemiller A, Andre K, Stein D, Blick G, Greenberg RN, Kinder C, Zolopa A, Binder-Scholl G, Tebas P, June CH,...therapy approaches to sickle cell disease. Hoban MD, Orkin SH, Bauer DE. Blood. 2016 Feb 18;127(7):839-48. ...

... a molecular leash for your protein. If you’re working with an uncharacterized protein, or a protein for...for antibody-based purification; has inherent enterokinase cleavage site GST Large Protein 26 Purification...context of a cell. FLAG tags can be cleaved by enterokinase, which recognizes DDDDK^X, cleaving after the...rounds of purification, with the cleavage reaction taking place between column runs. The TEV protease itself... Eric J. Perkins...

...Fujii: In the last several years, my lab has been working on development of technologies for biochemical ... molecules even enables us to skip the step of knocking-in. In 2013, we showed that isolation of a single...in the near future. Addgene: Has your lab been working on any other technologies that you would like to...that involvement in the scientific community is a kind of duty for researchers when considering the peer-review... in my lab then when they become independent. Looking for enChIP Plasmids? All Hodaka Fujii lab plasmids...

...that end, here are my personal 10 commandments for making it all work – most of the time. #1 Thou shalt find...happy at work, you will find balance. If you enjoy working and come home happy in the evening, your friends...of support to help with elder care, carpooling, cooking on nights when you can’t get home, babysitting ... really hard not to do this. For a family with working parent(s), the sooner your kids can make a decent...(except I am glad she never told me how much hitchhiking she did in Israel until after the fact). #9 Thou...

...cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching...effective and with no additional off-target edits, making them ideal for size-limited AAV delivery. PE6c ..., et al. (2023). Dynamic mapping of proteome trafficking within and between living cells by TransitID.... bioRxiv – doi: 10.1101/2023.02.07.527548.    Tracking cellular history with protein ticker tape By Brian...call for thin sections of tissue, the Wu Lab is working to create a three-dimensional atlas of protein ...

...amazing 111 countries (Figure 1)! But while we were making the materials available to many countries, import...stays in transit for a certain number of days and tracking average transit time for each courier we contracted... to increase accessibility to our repository by making it easier to find materials and learn about them...information are truly accessible, not just available.   Looking forward Over the past twenty years, Addgene has... accessible to scientists across the globe?” By asking this question, and by listening to the answers ...

... used for protein localization, isolation, and tracking experiments (Rizzo, 2009). With many different...aggregation and cross-react with anti-GFP antibodies, making them difficult to use in a multicolor/multichannel...fluorophore-labeled antibodies with very specific reactivities making them more useful for multicolor imaging experiments...Davidson, and David W. Piston. "Fluorescent protein tracking and detection: fluorescent protein structure and...Blog Rabies and neuronal tracing Retrograde AAV: Making the journey from axon to nucleus AAVs CREATEd for...

...make sure your plasmid has a promoter suited to working in your host organism. Because transcription machinery... and there is very little crossover. Generally speaking, promoters in bacteria are less diverse and complex...elements, insulators, and silencers; however, some "leakiness" of transcription may occur. This is normally ...expression mRNA Mammalian promoter from phosphoglycerate kinase gene.  Constitutive  Widespread expression, but...optogenetics mRNA  Ca2+/calmodulin-dependent protein kinase II promoter Specific Used for neuronal/CNS expression...