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... poking around on Addgene’s Fluorescent Protein Guide to In Vivo Imaging, you’ll pretty quickly notice...fluorescent proteins to observe what’s happening inside cells for at least a couple of decades. Green is...jellyfishes and corals, but all of them fluoresce outside the transparency window of mammalian tissues,” ...capable of revealing, for example, how muscles work inside a living animal. For those making use of these ...

...themselves. Therefore, your cloning strategy needs to identify cells containing the plasmid construct you’re ...positive selection strategy, here are a few to consider. Antibiotic selection Selection is straightforward...backbone contains your gene of interest. Auxotrophy Besides antibiotic selection, you can select for a successful...biology reference Watch molecular biology protocol videos ...

...software. Vectr has layers, which let you lock and hide individual vectors (shapes defined by 2D points ...OS X, and Linux. Inkscape is versatile and has a wide selection of tools for drawing and editing shapes...which requires users to give appropriate credit, provide a link to the license, and indicate if changes ... for reuse with modification. It’s always a good idea to double-check the image’s license information ...

...biggest advantages of such a tool is being able to identify specific transducer complexes that are activated...was more challenging. The Roth lab used structure-guided protein engineering and experimental refinement...protein signal transducers. The TRUPATH collection provides scientists with a toolkit to gain an understanding...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...

...This is where affinity tags come in – they are peptides which do not naturally exist in most expression...antibody is an alternative to amylose resin and also provides a visual detection mechanism via applications ...clean up step of the tag – a handy feature. If you decide not to cleave the GST tag after/during purification...Addgene.org Addgene antibodies Addgene’s antibody guide Antibody protocols  Resources on the Addgene blog...

...CAST, the nuclease-deficient CRISPR-Cas system identifies and binds a genomic target using a gRNA, and ...for a variety of loss-of-function mutants could provide a streamlined, one-size-fits-most approach. evoCAST...Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https...Additional resources on Addgene.org Addgene's CRISPR Guide (Jump to Large Edits) CAST Plasmid Collection ...

...these subpages, our updated CRISPR Guide and CRISPR History provide a great overview of the development...characteristic probably explains why pC016 - LwCas13a guide expression backbone with U6 promoter has already...member Alex Chavez) Day 11: Pooled libraries: Genome-Wide Screening Using CRISPR/Cas9 and Plasmids 101: NGS...

...short enough that everyone stays on the same page. Ideally, these meetings include all collaborators, from...meeting has an equal opportunity to contribute. Using video conferencing tools like Zoom or Google Hangouts ...pace at which problems can be addressed and new ideas generated. Furthermore, this casual communication...and grant applications in real-time so they can provide input at the times that work best for them. This...

...deposited at Addgene enhance scientific sharing worldwide but a select few are particularly special. Over...molecular tools with the scientific community worldwide, making it easier, cost effective and faster; ... Currently, there are roughbly 400 depositors worldwide who have one or more blue flame plasmids, with...literally enabled sharing with 100's of investigators worldwide. Now everything from every paper is routinely ...

...Molecular cloning techniques are some of the most widely used techniques in the biological sciences and ...unwanted mutations due to excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific...during PCR or any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations...Amplification efficiency is highest when using high fidelity DNA polymerases like phusion. An 18-cycle PCR ...

...Career: Doer, Manager, Leader” by Charlotte Beers, divides workers into three cycles: Doers, Managers and ...motivate their teams, it is the leader’s job to provide the driving inspiration, to create a productive... Understanding these three roles allows you to identify what you need to do to be successful in a specific...bringing and effecting solutions to both known and unidentified issues. There are many good resources on the...

...their work.  Check out Joanne's Reddit AMA   Guidelines for giving feedback Use formal and informal ...real change is possible in your eyes so their confidence is restored. A good manager does not hold grudges...best? What 3 things worked least well? Manager provides feedback (may be the same as self-assessment or...about real performance issues. Watch this little video to learn more about indirect language from Steven...

...quick look at your order status on addgene.org identifies the hold-up. Your Material Transfer Agreement...quickly! If your TTO receives a Golden Stopwatch, consider sending an email to thank them--after all, they...unprocessed agreements slow down your research; provide specific examples if possible Tell them about the...exchange of intellectual property (as inventions, ideas, plasmids) among universities and other research...

...know all about antibiotic resistance genes, let’s consider another basic element of any plasmid: the origin... intend to use, and whether or not you need to consider your plasmid's compatibility with one or more ...copy number varies. See below for additional considerations. *Requires pir gene for replication (reference... external factors contribute as well. These considerations are especially useful to keep in mind if you...

...Addgene closed for almost two weeks and with labs worldwide closing for unknown amounts of time, we were facing... enhances productive collaborations as well as provides visibility for grant-driven research being done...each of many grantors supporting research could provide Addgene with a small amount of funds, the community...and projects to serve the needs of scientists worldwide with Addgene’s traditional excellence through ...

...what gets packaged into the AAV molecule. What’s outside of the ITRs does not get packaged in the AAV vector...the WPRE. TL;DR? Watch our AAV Transfer Plasmid video!   Now you’ve got a basic understanding of some...Lisowski L, Alexander IE, Logan GJ (2019) A User’s Guide to the Inverted Terminal Repeats of Adeno-Associated...basic viral vector elements Read about four considerations for the perfect AAV tool Learn about synthetic...

...explaining a technical piece of research to others outside that specialty, who may be other scientists, or...journalists, or policy makers. The term editor covers a wide variety of careers and jobs. At Science, which is...financially critical. Those who work on the publishing side of science have professional meetings, societies...in the science communications world, you might consider an internship or a short program to build skills...

...Taylor, 2014). They’re so simple that we can’t even decide if they’re alive or not. Yet these simple, small...Virus structure While all viruses have a genome inside a protein shell known as a capsid, some also have...march to the beat of their own drum. They have a wide variety of possible genome types that can be either...copies, which are released from the cell like baby spiders from an egg sac. (Okay, some enveloped viruses ...

... the literature and find an ideal antibody against Protein A and an ideal antibody against Protein B. ...specificity to the target while constant regions are identical for antibodies within certain classification groups...with human antibody sequences.  Additional considerations Although isotype conversion is routinely used...

...attention and enjoy anecdotal forms of information — identifying with a character, building emotional investment... do and should tell stories all the time. They provide accounts of their research struggles in papers.... a weekly lab meeting as you’ll need to for an outsider. And you won’t need to dive as deep into your ...preprint servers, share your email address far and wide, keep your website up to date, invite people to ...

...hybridomas) that secrete antibodies in culture and provide renewable reagents with reliable and reproducible...first NIH grant that Addgene has received. Addgene prides itself on being financially self-sustaining through..., Addgene’s interim Executive Director. “NABOR provides a new model for the creation and acquisition of... enable researchers everywhere.” The timing is ideal as the NIH’s BRAIN Initiative has been funding laboratories...

...lysosome, and degradation of autophagic substrates inside the lysosome. Historically, methods for studying...dynamic process into a static measurement and it provides limited information about what materials or organelles...autophagic activity. The aim of this post is to provide an overview of four autophagy biosensors currently...protein plasmids and resources Fluorescent protein guide: Biosensors ...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...Article contributed by Shreya Vedantam CRISPR/Cas9 is widely used genome editing tool. However, Cas9’s endonuclease...when studying neuronal activity. The researchers identified MerMAIDS from their analysis of different publicly...comprised of thousands of loops. These loops not only provide a 3-D structure for packing genomic DNA into nuclei...

...fusion protein, etc...).  There are many online guides – including an excellent series of posts by Joachim... Addgene blog – outlining various important considerations when choosing a FP, but much of the primary...for, the Table of Fluorescent Protein Properties provides a master list of all proteins in the database,...try out the site and contact us if you have any ideas or requests.  Or, if you have experience in Python...

...advancement of SARS-CoV-2 research because they provide rapid access to validated reagents, especially ...distribution of cell lines from repositories on opposite sides of the world shows that science is truly a global...dedicated facilities and manpower that a repository provides. Repositories around the world facilitate the ...15562-9 Subbarao K, Roberts A (2006) Is there an ideal animal model for SARS? Trends in Microbiology 14...

...performed with embryos and adult fish. Zebrafish are an ideal model organism for these experiments because developing...cell death was confirmed by TUNEL staining.  The video below shows what happens to the zebrafish’s heartbeat...circulation of following FAP-TAP light activation. Video: https://www.youtube.com/watch?v=xOtH5bs4HiI Advantages...Resources on Addgene.org Visit Our Fluorescent Protein Guide Page Check out MiniSOG2 and SuperNova, two other...

...that we could discuss our projects and ideas every day and decide on the best way to structure roles and...-group meetings to discuss day-to-day progress, ideas, and ways to improve the lab. “An effective working...including their "Making the Right Moves: A Practical Guide to Scientifıc Management for Postdocs and New Faculty...

...immunoprecipitation (ChIP) is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works...up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires...ll need to ensure you have a bead-only control alongside your input control. Elute the DNA after washing...you generate your data. However, it may be a good idea to add "patience and an extra-methodical approach...

...addgene.org Addgene's Antibody Guide rAb Affinity Purification Protocol Video rAbs in the Addgene repository...to upregulation or downregulation are easier to identify by comparing peak positions to the base expression...2. Quantification using MFI makes it easier to identify and study these subtle shifts. Additionally, you...

...license. Better performance = better data In side-by-side tests with popular red and yellow GECIs, OCaMP... while red indicators jRGECO1a and jRCaMP1a are widely used as well. To complement these tools, we recently...red.  However, current red and green GECIs aren’t ideal for two-photon imaging with newer lasers in the ...

...lentivirus, retrovirus, AAV, etc.). When you’ve decided on an application, our Viruses as Biological Tools...adjustable nature of viral libraries makes them the ideal vector for the job. Therapeutic applications Vaccine...popular nucleic acid-based vaccines. These vectors provide several advantages over traditional vaccines, including...and cell-specific targeting features make them an ideal delivery system for human patients. Multiple viral-based...

...this work? Through molecular biology teamwork. The guide RNA (gRNA) specifies the editing target site on ...deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10 version with a faster ...enzyme. After testing numerous TadA variants, they identified TadA-8e, which edits 590-fold faster than TadA... base editors for your research Read our CRISPR guide Find plasmids for your research ...

...researchers.There is both theoretical and empirical evidence (in psychology, cancer biology, pre-clinical life...) do you really appreciate how few details are provided in most published papers. The precise timing, ...code, and other materials. The use of reporting guidelines can also help remind us to include all of the...funders in the Transparency and Openness Promotion Guidelines; preregistration to increase clarity in study...

...relationships than at scientific conferences. Conferences provide the opportunity to meet people who are interested...larger your profile in the community Job hunting outside of academia is all driven by personal connections...you are spending on a conference? One thing to consider is the size of the conference. Large conferences...scientists at your level of training is always a good idea. Make contact with other attendees in advance. Join...

... more confidence in gene expression readouts from assays such as qPCR or microarray. But, aside from facilitating... replication are initiated before the cell has divided. This means that genes located near the origin ...engineering, the expression of enzymes in a pathway will ideally be optimized to promote generation of the desired...

...Visit our viral vectors guide Find our chemogenetics and optogenetics science guides ...tissue culture dish but can also happen in real-time inside a mouse’s brain.  But the brain is a tricky organ... Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin engineering enables minimally ...

... the Addgene website Promoter Guide CRISPR Guide Lentiviral Guide  ...Tagging relevant proteins with a degron tag can provide inducible protein degradation. These systems work...

...Addgene Resources Cre-lox Resource Guide Fluorescent Protein Resource Guide Fluorescent Protein Blog Posts...single heavy chain, and they are small and stable inside cells. Nanobodies binding GFP were designed in ...these fusions retained the ability to bind GFP. The idea of using GFP as a scaffold suddenly seemed very ...

...translational termination features with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette...knockout – conversely, putting the loxP sites on either side of a gene (called “floxing”, for “flanked by loxP...experimental constraints. This has been used for a wide range of purposes, including activating an oncogene...switch recombination at individual switch regions evidenced through Cre–loxP–mediated gene targeting. Gu, ...

...respectively. Primary cell type Gene targeted by guide Editing efficiency hiPSCs  EMX1  67% Mouse BM...in cells electroporated with RNPs using the same guide. Nanoblades also had lower off-target effects when...insertion of a DNA sequence ranging from a single nucleotide change to large insertions like the addition ...Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources Learn about viral...