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...twitter @Vopinator.     References: Halpin-Healy TS, Klompe SE, Sternberg SH, Fernández IS (2020) Structural...Insertion of the mini-transposon can occur in two different orientations, but is strongly biased towards T-RL...combined in single-plasmid INTEGRATE (pSPIN), which offer simple transformations for smaller cargos, and the...integration efficiencies achieved with VchINT, at two different incubation temperatures. Data adapted from Vo ...

...denotes intersection and the – operator denotes set difference. That’s the intuition for how genome math helps...enhancers from step 2. See which ESCs die or differentiate Step 3 reveals which NANOG-related genes are...distinct enhancer disrupted. As our ESCs die and differentiate over time, we periodically use next-generation...Therefore if a gRNA drops or disappears over time, we infer that the enhancer it targets is a “pillar of function...by starting with all of the DHS sites from 125 different human cell types from the ENCODE Project. DHS ...

...viral infection), producing firefly luciferase. Firefly luciferase can readily be measured to quantify...single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific gene start...to transcribe the reporter gene (e.g. firefly luciferase). The polymerase recognizes the gene start signal...transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula J., Stefan Finke, and Karl-Klaus...

...limitations associated with SpCas9. Cas13a (previously referred to as C2c2), has several unique properties that...for target recognition and cleavage). C2c2 (now referred to as Cas13a) is unique in terms of structure ...remainder of this blog post. Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds ...SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus, genotype human DNA, identify...Gearing contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...

...human pluripotent stem cells By Jennifer Tsang CRISPR gene interference (CRISPRi) and gene activation (...For labeling the plasma membrane, they tested different target sequences for improved selectivity and ...into the supernatant. Four plasmids containing different nanobodies (NB) were generated in pMAK:  pMAK461....1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR...

...moving around to explore the environment has a different motivation (and brain areas associated) compared...addiction. Anxiety tests Rodents have a natural preference to stay in dark areas (protected) as opposed ...to brightly illuminated ones (risky). They also prefer to explore novel environments by initially moving...anxiety. On the other hand, the control group preferred staying in the dark compartment (Howerton at al...these in a  future blog post, so stay tuned!  References Alpár, Alán, et al. "Hypothalamic CNTF volume...

... to determine the pros and cons of using many different types of gRNA design software. When you are happy...inserted or deleted. Each lab will use a slightly different method to genotype their pups. Taking a toe, a...weeks old and use the ear tag method (creating different patterns of ear holes and using that material ...latest and greatest in scientific content.      References 1. Cho, Seung Woo, et al. "Analysis of off-target...plasmid into zygotes." Development, growth & differentiation 56.1 (2014): 122-129. PubMed PMID: 24372541...

...interviews, or get several interviews but no job offers, is a failure to clearly articulate one's talents...to convince me that their search committee is different and needs to be told all of the nuances of the...technologies, i.e. video interviews. For point of reference, I submitted my applications all by snail mail...hoppy pint of IPA or a few drams of single malt (preferably from Islay or Skye). Good luck with your search...University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz at the National Institutes...

...delivering scFvs that inhibit HIV.   References and Resources References: Ahmad ZA, Yeap SK, Ali AM, Ho WY...for high affinity scFvs. Bound phage are then proliferated by infecting bacteria for additional rounds ... high affinity binding. Bound phage are then proliferated by infecting bacteria for additional rounds ...science.3140379  Bradbury ARM, Sidhu S, Dübel S, McCafferty J (2011) Beyond natural antibodies: the power...

...labs. While many labs approach the situation differently, some adopting simple note-taking tools, others...functionalities with experiment records. Many different types of ELNs are available on the market today... comparison to paper-based record keeping, ELNs offer functionalities that are hard to imagine on paper...desired functionalities i.e. main priorities of different ELN features, ranging from whether respondents... allows you to assign specific permissions to different lab members to avoid any issues later on. Do you...

...C-terminus of the fluorescent protein. I prefer to describe the different parts of the fusion from N- to C-terminus...tagged protein because of its size. This type of interference is known as steric hindrance. For instance, ...advisable to generate multiple constructs with different insertions, since it is difficult to predict which...can follow him on twitter: @joachimgoedhart.   References Adjobo-Hermans, Merel JW, et al. "Real-time visualization...

...of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded guide RNA (gRNA...agrobacterium-mediated delivery. Summary While different in many of the specifics - promoters used, precise...mediated genome engineering in plants isn’t all that different from how it’s used in other systems. Luckily, ... don’t be afraid to try your hand at CRISPR. References Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov...of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol...

... Even low-frequency editing could make a huge difference in DMD, and it’s estimated that exon skipping...respectively. Although each study used slightly different methods, they each observed the same positive ...disease type C. Although the three DMD studies referenced here represent a great step forward for CRISPR...many distinct CRISPR therapies to match these different mutations. For each therapy, effectiveness and...treatments may also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular...

...bottom of this post for a basic synopsis of the differences amongst the Brainbow systems. Pre-Brainbow techniques...to the promoter will be expressed. This system differs from Brainbow-1.0 in that transient Cre recombination...needed to mark individual neurons, the system also suffered from a number of limitations. First, the imaging...controlled by FLP/FRT recombinase. When placed under different promoters, Brainbow and Flpbow can be used to ...2.1 plasmids Brainbow 3.0, 3.1, 3.2 plasmids References: Transgenic strategies for combinatorial expression...

...PiggyBac transposons, the Woltjen Lab compared different variants of the polycistronic cassettes and discovered...a donor in FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy...is a new member of this class that works via a different mechanism - the PhoCl protein is irreversibly ... are other RFP variants available that may be preferred for their intended uses, they provide a well-defined...of essential genes in vivo. Utilizing CRISPR interference (CRISPRi) technology, the arrayed knockdown ...defined domains can be swapped onto other proteins to confer functionality. In Narayan et al., they used this...

...Szymanski in January 2016 in Viruses (5). Please refer to the paper for more details on the assay, including...however, a physical fragmentation strategy is preferable. Nebulization involves forcing DNA through a ...on the membranes will then be exposed to a lysis buffer to release recombinant proteins directly onto the...then suspend a freshly harvested dose of cells (preferably in log phase) in blocking solution (around 108...thinking. Find her on Twitter @JessicaSacher.   References 1. Nobrega, Franklin L., et al. "Revisiting phage...

...“NCBI Site Preferences” box. Once on the Preferences page, navigate to “PubMed Preferences” and click ... that you know how to navigate to the PubMed Preferences and Filters pages, be sure to check out the other...

...Request service, which offers over 3,000 eligible transfer plasmids and five different serotypes. Not sure... pAAV_hSyn-PdCO-mScarlet-WPRE AAV1 Optogenetics Ofer Yizhar New viral tool pAAV-Ef1a-fDIO hChR2(H134R...

...modified allele is selectable, it can be transferred to a different genetic background via P1 transduction...Figure 2). In the following sections, key steps that differ from traditional restriction enzyme cloning will...could be used to move the modification into a different background, if needed. This approach is best suited...science communication and in vitro diagnostics.     References 1. Costantino N, Court DL. Enhanced levels of...

...AAV constructs CRISPR interference in Candida albicans A new CRISPR interference (CRIPSRi) platform for... to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida albicans E. coli genome-wide inhibition... Jennifer Tsang...

...animals’ skeletal muscles led to another clue: differences in the kynurenine pathway of tryptophan degradation... skeletal muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into...Get started with PGC-1 plasmids from Addgene. References: Skeletal muscle PGC-1α1 modulates kynurenine...M, Bhat M, Schuppe-Koistinen I, Pettersson AT, Ferreira DM, Krook A, Barres R, Zierath JR, Erhardt S, ...

...available at Addgene CRISPR-Cas9 References, Protocols, and more References: Generation of Gene-Modified ...the monkey story hit the presses, another very different bit of CRISPR news was circulating. Scientists... work. Last month, the Lawrence Berkeley Lab's Jennifer Doudna and Eva Nogales presented the first detailed...

... online. A few examples are provided here for reference: Untergasser Lab Preparation of chemically competent...use the Mix & Go! E. coli Transformation Kit and Buffer Set  from Zymo Research to make competent cells...then wash and resuspend the cells in the provided buffers. The competent cells are then aliquoted and stored...protocol)  Cells prepared using the Z-competent TM buffer can be easily transformed without a heat-shock ...

...finding the best way to organize your time. At a conference, you could be honing your science communications...that lab and interacting with the lab members.”- Jennifer Tsang, Science Communications and Marketing Coordinator...Addgene blog Read all careers blog posts Read our transferable skills guide Resources on Addgene.org Find... Jennifer Tsang...

...shipped over 350,000 individual plasmids to 5,000 different research institutions. This has given us a unique...by the influx of plasmids for reprogramming differentiated cells into pluripotent stem cells. Plasmids...HA-Ubiquitin-WT and HA-Ubiquitin, and we now have 19 different ubiquitin plasmids in the repository. 100,000 ... and modified by so many labs and for so many different purposes that we had to create a CRISPR page to...

...the researchers applied the LITE system to 28 different genes to establish it as “a novel mode of optogenetic...activators are easy to expand or combine with different effectors. I’d encourage people to get creative...apply LITEs for the transformation of neurons, she offers a tip: save yourself some time by making an AAV1...readers, how will you get creative with LITE? Reference: Silvana Konermann et al. "Optical control of ...

...inflammatory cytokines - interleukin (IL)-12 and interferon gamma (IFNγ) - and eliminate cancer cells. 2....physiological and morphological changes. Tumors differ in their genotypic and phenotypic identities based...by cancer. As mentioned in this article, cancers differ in practically every facet starting from manifestation...manifestation to their specific interactions with different cell types. A thorough understanding of the genotypic...interested in exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in ...

...York City, NY, USA 40.7128°N 74.0059°W – Deanna McLafferty I’m a New Yorker, so it’s not easy to convince...myths that abortions cause breast cancer and infertility or to argue against unnecessary clinic regulations...and fiction. Our health depends on it. Deanna McLafferty is an editorial assistant and activist. Washington...enough.  Nashville, TN, USA 36.1627°N 86.7816°W- Jennifer Stancill I am a research biologist by training...supporters is much larger and stronger that I expected. Jennifer Stancill, Ph.D., is currently a post-doctoral ...

...simplifies your viral vector options. Almost any AAV transfer plasmid in our repository can now be packaged ...packaged on demand in five distinct serotypes, offering you unparalleled flexibility. But this is just the beginning...developments? Subscribe to the Addgene newsletter. References Ayoubi, R., Ryan, J., Gonzalez Bolivar, S., Alende...A., Trimmer, J. S., Rego, M., Freedman, L. P., Ferrara, F., Bradbury, A. R. M., Cable, H., & Longworth...

... and Negative Selection for Plasmid Cloning by Jennifer Tsang There are many strategies for finding your...Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi by Jennifer Tsang Learn about mNeonGreen, mTFP1, and mWasabi...Precision and Flexibility to CRISPR Editing by Jennifer Tsang Prime editing is a new CRISPR technique... Jennifer Tsang...

... might not fold well, you should screen a few different vectors to see which gives you the most functional...two sets, each of which includes plasmids with different varieties of tags, both N- and C-terminal. Each...improve folding and disulfide bond formation. If one prefers expression in E. coli, the use of a thioredoxin...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system...

.... The following Friday, I received the official offer. I was so happy to hear back, I accepted that day... food service industry, which followed a very different schedule. Fortunately, I was provided with a detailed...gave me enough time to become familiar with the different types of issues that were important to this function...Specifically as a co-op, it is encouraged to explore different teams and work on projects there. At this point...

... value, clearly communicate exactly what a site offers to their users, and most importantly, allow those...get scientists excited about what Addgene has to offer, and to help scientists find what they need - whether...wanted to highlight our blog, daily updates, and offer better exploration throughout the site.  Blugene...2011, and while you may have seen our mascot at conferences, on our website, or in social media, we wanted...

...plasmids for expressing different types of rhodopsins and sensors as well as references and links to other ...using both its Addgene ID number as well as the reference for its original publication. To help facilitate... citation that you can put straight into your reference section. Why do this? Not only does citing plasmids...

...the future. Studies show that the majority of job offers arise as a result of existing professional (and...interest, even excitement, in a position. Everyone prefers to hire someone that will be excited to be there...resume should also be customized to highlight different skills and to get the important information in..., you will never get the help that can make a difference. Having your resume forwarded personally gives...

...eyes to the many different jobs associated with science, especially if the conference is a big multidisciplinary...curious and interdisciplinary.   3. “There are many different ways to succeed – as loner or gregarious;  dictator...leaving academia should present their research at conferences to obtain a “crash-course in all the real life...

...nontarget strand. Protein Cas: Different CRISPR systems involve different proteins, but many of them are...laboratory applications. At Addgene, we sometimes refer to the programmable guide sequence as the spacer...scaffold. gRNA: "Guide RNA" is a general term referring to the RNAs used by Cas proteins, either a crRNA...

...the discoveries yet to come.      References and resources References Arbabi-Ghahroudi M (2017) Camelid...become appealing candidates for the detection of different cancers (Jin, et al. 2023).  So where do all these...    Figure 2: Genetically modified mice offer an alternative method to producing single-domain...

...general primer design, as well as primer design for different cloning applications. Primer3 and Primer3Plus...calculate how much insert and backbone you need for different types of cloning methods, such as restriction ... any cloning method. You can select from five different insert:backbone ratios. In-Fusion cloning molar...Resources on Addgene.org: Read our Molecular Biology Reference Find molecular biology protocols Browse our protocol...

...nomenclature throughout the rest of this blog post, GFP refers to sfGFP-GB1.   Tanenbaum et al. examined the power...SunTag? Tanenbaum et al. performed a variety of different experiments in their paper, and so can you! In...we’ll save that application for another day! References 1. Tanenbaum, Marvin E., et al. “A protein-tagging...Visit Our Fluorescent Protein Guide Page Find Luciferase Plasmids ...