...succesful scientists have time for fun and life outside of the lab? This final post in the PI Interview...other. The Church Lab enjoys each other’s company outside of the lab by sharing meals, going on trips, and...school year winds down, take some time to have fun outside the lab! How does your lab celebrate each other's... fun outside the lab...

...base editor, BE3, induces a low frequency of genome-wide off-target DNA editing. Using several rapid and ...-effective screening methods, David Liu’s lab identified and validated CBEs with reduced Cas9-independent... tool from Albert Cheng’s lab that enhances RNA-guided methylcytosine editing by TET1 by co-delivering...sheet Resources on Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited...

...aware of careers outside of academia. How do PIs assist their mentees who have goals outside of the university...anti-tech or pragmatic.” Advice for pursuing careers outside of academia Recently, graduate students and postdocs...skills that are applicable to a variety of careers outside of academia. She has had previous graduate students...

... 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 ...

...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...

...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...

...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 ...

...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 ...

...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...

...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...

...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...

...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...

...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 ...

...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 ...

...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...

...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...

...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...

...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...

...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 ...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

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

...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 ...

...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, ...

...CRISPR Background with Our Guide Pages Other Links Check out Pawluk et al's Video Abstract Read up on All...off-target edits of base editors, which convert a nucleotide base pair to another base pair at a specific .../35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic...

...measure the tension in the helical structure of spider silk, or the activity of a protein in a synapse... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...As the choice of fluorophore pairs is a key consideration for a FRET experiment, the possibilities can...PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein FRET Pairs." Sensors 16.9...

... are considered large deposits and are exceptions to our standard workflow.  Table 1: Guidelines for the...Plasmids with Addgene” blog post for a thorough guide to our online data entry process. Figure 1: Begin...application of the plasmid and will help a scientist outside your field understand if this plasmid will work...

...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...

...rescued by zygote co-injection of Cas9 and a single-guide RNA targeting the mutant allele. When all was said...hATTR) hATTR is caused by a variety of single nucleotide polymorphisms (SNPs) in the TTR gene and is molecularly...Off-target effects are also important safety considerations for all CRISPR therapies, but especially concerning...CRISPR therapies have made significant clinical strides since we first wrote this blog post. The future...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ... cells that make up the human body and G-baToN provides versibility in tracking and modulating the interactions...libraries encode target sites flanked by randomized nucleotides in place of a protospacer adjacent motif (PAM... viral aliquots from our plasmid collection to provide ready-to-use viral preps. Here are some of the ...

...bigger movement in a variety of research sectors to provide open source technologies and spaces where interested...One of the aspects of this project that makes it ideal for makerspaces is the data is freely available,...projects). This allows the project to be open to a wider group of people. This is a perfect starter project...equipment becomes obsolete amazingly fast, but with a wide range of skills among our members we’ve managed ...

...powerful than it is. The nuclease domain of Cas3 is considered weak, especially compared to the power of Cas9...Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394...Liao, M., & Ke, A. (2018). Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science, 361...CRISPR-Transposons Additional resources on Addgene.org CRISPR guide Cascade-Cas3 Plasmid collection ...

...There are several facilities nationwide where you can obtain excellent quality, high titer rAAV (including...certain in vitro and in vivo applications, you can consider using unpurified AAV from the supernatant (Goodwin... in precision and variability, qPCR is the more widely used method due to the accessibility of instruments...101 series!  This post was originally written by Didem Goz Ayturk and updated by Ina Ersing in August 2023...

...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap) genes...primarily found in the culture media, you should consider performing PEG precipitation. Instead of sonicating... details. Pro tips Watch this AAV purification video! It didn’t exist when I did my first iodixanol gradient... gradient purification, but I wish it had. The video gives a brief overview of how iodixanol purifications...