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We narrowed to 810 results for: nin

Showing: 101 - 150 of 810 results

  1. A Control for All Seasons

    Type
    Blog Post
    Published
    Oct. 5, 2023, 1:15 p.m.
    ...Such species-on-species staining can be partially addressed with stringent staining protocols and the use...the process could make it difficult to obtain meaningful results.  Biological replicates are distinct ...antibody specificity. B, secondary antibody-only staining will allow you to see nonspecific interactions...incubated with its antigen or a small epitope-containing peptide to block the antigen-binding site. The...      Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity must...inflammation. This tends to be most problematic when staining cells or tissues that are the same species as ...secondary antibodies, and labels omitted. Any signal remaining is autofluorescence and should be considered in...

  2. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    Published
    Aug. 9, 2022, 1:15 p.m.
    ...a lab we discuss the results together, explaining our reasoning to determine if the antibody is negative...cannot be completed. A 2015 review analyzing and explaining the antibody crisis states that up to 50% of ...Initiative Cell Census Network, the Wu lab will be screening over 3,000 antibodies for use in mice and human...consistent antibodies for specific markers. The Screening Criteria While choosing which antibodies to screen...uses. We therefore use both rAb and mAB in our screening. To screen the antibodies, we have developed ...to see the reproducibility and accuracy while screening. We also compare different clones of the same ...multiple people in the lab conduct “independent screening”, where they look at the same section and determine...

  3. Pooled Library Amplifications

    Type
    Blog Post
    Published
    May 30, 2024, 1:30 p.m.
    ...Protoc. 2017 with modifications Screening pCMV-Rep78/68 Scanning Saturation Mutagenesis (SSM) Pooled...amplification protocols tend not to work as well.   Defining your library First, you’ll need to collect specific...aim for somewhere between 30x and 200x coverage, meaning that you need 30 to 200 times as many colonies ... be in direct competition with other colonies containing smaller, recombined backbones that may replicate...Re-transformation of the SPLINTR Libraries (PDF, 401 KB) Screening Zhang Lab - Multiplexed Overexpression of Regulatory...Amplification and Sequencing Protocol (DOCX, 32 KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated...-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab, and the Broad...

  4. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    Published
    Dec. 17, 2020, 1:30 p.m.
    ...variable amounts of experimental manipulation and screening.  A co-selection strategy for CRISPR editing in... with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach detailed...low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering rare editing events...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal mutation...to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of lethal ...parental animal, are rescued progeny, which makes screening extremely rapid. This stringent selection allowed...al., 2014), inactivation of non-homologous end-joining results in a further increase in knock-in efficiency...

  5. Healthcare Consulting: A Door to the Business of Life Sciences

    Type
    Blog Post
    Published
    Oct. 13, 2016, 2:30 p.m.
    ... up a large and diverse group of professionals running the marketing, commercialization, collaborative...client might need help with the intricate task of designing a strategic vision for a portfolio of candidate...edge; a consultant might outline the impacts of combining targeted molecular therapies for cancer with chemotherapies...from previous consulting experiences, educational training, and conferences, and synthesizing this knowledge...externally to clients and experts. Hence, active honing of communication skills is a must to be successful...have greatly benefited from and appreciated the training I’ve received in concisely and accurately communicating...and that they can be ameliorated by thoughtful planning and engagement, leaving mental energy to enjoy...

  6. Plasmids 101: Antibiotic Resistance Genes

    Type
    Blog Post
    Published
    Jan. 30, 2014, 3:29 p.m.
    ...resistance as a marker for successful cloning. In these instances, the cloning vector typically has two separate...Posts Learn How to Use Blue White Screening to Simplify Your Cloning Experiments See Where You Can Find...cells on average! Without some means of quickly determining which cells successfully received the correct... from the bacterium's perspective – a plasmid-containing cell must replicate the plasmid in addition to...it allows a scientist to easily detect plasmid-containing bacteria when the cells are grown on selective...gene and acts as a marker for the mutation. When designing these types of experiments it is best practice... or completely removes (in the case of Gateway cloning) one cassette. Counterselection allows the scientist...

  7. Early Career Researcher Toolbox: Free Online Molecular Biology Tools

    Type
    Blog Post
    Published
    Sept. 12, 2023, 1:15 p.m.
    ...types of cloning methods, such as restriction enzyme cloning, Gibson assembly, or In-Fusion cloning.  NEB...Design primers for Goldengate cloning   Design primers for In-Fusion Cloning Design primers for Gibson Assembly... any cloning method. You can select from five different insert:backbone ratios. In-Fusion cloning molar...design, as well as primer design for different cloning applications. Primer3 and Primer3Plus: For general...sets for high complexity Golden Gate Reactions Cloning ligation reaction calculators These links can help...

  8. Plasmids 101: What is a plasmid?

    Type
    Blog Post
    Published
    April 2, 2020, 6:17 p.m.
    ...Allows for selection of plasmid-containing bacteria. Multiple Cloning Site (MCS) Short segment of DNA... of cloning methods (restriction enzyme, ligation independent, Gateway, Gibson, etc). The cloning method...into. Regardless, once the cloning steps are complete, the vector containing the newly inserted gene is...an origin of replication, selection marker, and cloning site. The ease of modifying plasmids and the ability...applications. Some common plasmid types include cloning plasmids, expression plasmids, gene knock-down ...Reference includes information about molecular cloning, how to choose a plasmid vector, molecular biology...

  9. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...transitions are edits between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine...gene editing. Cytosine base editors (CBEs) and adenine base editors (ABEs) chemically modify target bases...included in the CGBE. They also developed a machine learning model capable of predicting editing outcomes of...sometimes processed by error-prone non-homologous end joining (NHEJ). To mitigate this, the team introduced the...of unwanted DNA edit. NHEJ Non-Homologous End Joining An error-prone cellular process to repair DSBs.... the first type of base editor invented. ABE Adenine Base Editor Converts A → G; the second type of ... screens, target-library analysis, and machine learning. Nature Biotechnology, 39(11), 1414–1425. https...

  10. Transferable Skills Guide: Time Management

    Type
    Blog Post
    Published
    Dec. 5, 2017, 1:52 p.m.
    ...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...undergrad working with cancer cell lines. As I started planning out my own experiments in graduate school, I had... into three conceptual chunks: prioritization, planning, and procrastination. Time management through ...one of them. That’s why, as I’ll discuss in the planning section below, it’s important to dedicate specific...splitting my cells first. Time management through planning I probably wouldn’t have fallen into my cell splitting...projects to operate this way now. Talking about “planning” is easy, but actually doing it in a productive... way can be quite difficult. In my experience, planning has 3 components: A mechanism for blocking off...

  11. Plasmids 101: Methylation and Restriction Enzymes

    Type
    Blog Post
    Published
    June 30, 2016, 2:30 p.m.
    ... methyl group to the adenine in AACNNNNNNGTGC or GCACNNNNNNGTT Impact on cloning and digests Although...naturally occur in bacteria for purposes other than cloning or confirming plasmids. It turns out that restriction...one or a few restriction modification systems containing methyltransferases that add methyl groups to ...DNA: Dam methylase adds a methyl group to the adenine of GATC stretches of DNA Dcm methylase adds a methyl...coli strain and will therefore have methylated adenines in any GATC sequences found in the plasmid. When...enzyme will cut? Whether performing a digest for cloning purposes or for diagnostics, we suggest double ...can ensure this site remains unblocked by first cloning your DNA into a dam–/dcm– strain of E. coli such...

  12. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    Published
    Feb. 10, 2016, 3:30 p.m.
    ...Read about Other Plasmid Cloning Techniques like SLIC and Golden Gate Cloning Learn How to Verify Your...same efficiency as Gibson Assembly and GeneArt cloning - but at a much lower cost. Read on to find out...an appropriate vector. Gibson Assembly, GeneArt Cloning, and SLIC can all be used to assemble multiple ...described above, you design two primers, each containing the desired mutations, to amplify both strands...strands of a plasmid template. In addition to containing the mutations, the primers also have some homology... head-to-head with Gibson assembly and GeneArt cloning, the results are favorable. With 17-bp homology... observed efficiency of mutagenesis (mutation-containing colonies/total colonies) is about 84% for each...

  13. Finding Your Perfect Job After University

    Type
    Blog Post
    Published
    Jan. 12, 2016, 3:30 p.m.
    ...system), in 2009 (at the beginning of the financial recession) I knew obtaining work in scientific research...Working in industry: Microbiology On returning from Ghana at the beginning of 2010, I explored options for ...physically carrying out experiments like PCR and running gels, but in research you also experience a lot...positive experience. I enjoyed helping people and learning to explain concepts in ways that people from another...looking for other career options after 6 months. Returning to university to study a Masters of Science I ... saved money from my role in Industry and took evening work to help cover the costs. My MSc courses were...Industry was really valuable; I had received all the training and experience relating to good manufacturing ...

  14. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    Published
    Dec. 20, 2022, 2:15 p.m.
    ...Positive Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the activator... herpes simplex virus. tTA binds to promoters containing TetO elements (often linked in groups of seven...change that prevents binding to the promoter and turning transcription OFF. Despite their bacterial origins...systems function well in mammalian cells, and TRE-containing promoters can be used in the repressible manner...activation domain VP16. cTA binds to promoters containing putative operator sequences (CuO) (linked in ...change that prevents binding to the promoter and turning transcription OFF. The cumate switch system functions...broad QF expression-induced lethality in flies. Riabinina et al. also developed chimeric transactivators...

  15. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    Published
    Nov. 21, 2019, 1:53 p.m.
    ...gets “Lexicon Training.” Since the lexicon is a “living” document, I update the training frequently. In...the confusion in our meetings. A lot of our LIMS planning involved “plates,” and our scientists and lab ...misinterpretation, that misinterpretation was definitely happening. Our solution for this problem was the creation...simplified the first weeks or months when I was learning the terminology of a new field. Annotate: When...we define new and potentially ambiguous terms. Defining words and applying annotation can be particularly... meeting is using the same words with the same meanings consistently, meetings will be shorter and more...Happier colleagues. Feeling like people aren’t listening to you or understanding you can be incredibly ...

  16. Addgene's Tips for Plasmid Quality Control

    Type
    Blog Post
    Published
    Jan. 14, 2016, 3:30 p.m.
    ...starts by determining what features you expect to differ between plasmids, and selecting or designing a primer...antibiotic resistance, restriction sites used during cloning, and most importantly, the predicted full plasmid...quality, as it will catch any errors made during the cloning, sample handling, or sequence assembly steps. All...on the number of features we need to verify. Determining which features to verify will depend on the particular...its predecessor. Important features may include cloning junctions (where most sequence assembly errors ...mutations, in-frame protein fusions, and multiple cloning sites. In the simplest case we have a standard ...you design your custom primers. Remember, when designing a sequencing primer, you need some space between...

  17. Story of a SynBio Startup: RevBio's Epiphany (or Lack Thereof)

    Type
    Blog Post
    Published
    June 5, 2014, 3:32 p.m.
    ...something, even if that thing is opening a pizza joint. Silly ideas (mining local salt deposits to sell at...with a similar conundrum – she had the skills, training, and motivation, but there very few jobs available... journal clubs outside of work. Our scientific training was put to use as we explored ideas, planned out...” We kept our day jobs and worked weekends and evenings on the project, but still continued to generate...and another $30,000 in mentorship and business training. The money is a drop in the bucket against the... the technology we will need, and the business training to make the most of it. It has also allowed us...and beautiful, to engage with the public in a meaningful way about science, and to have a job that we ...

  18. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    Published
    Oct. 8, 2024, 1:15 p.m.
    ...just for lab specimens — there is evidence that lightning can also promote gene transfer in soil bacteria...resistance-mediated selection is indispensable for cloning work, antibiotics and plasmid-encoded resistance... in the lab but also causes outbreaks of food poisoning, the difference is virulence plasmids! This category...natural inducible expression systems that allow tuning of metabolic pathways. Fertility (F) plasmids: ...2024), and others have proposed developing new cloning techniques based on these vectors (Shareck et al...have more than one function: for example, Gateway cloning relies on genes found on an E. coli fertility plasmid...this F plasmid is critical for successful Gateway cloning. In addition, scientists have engineered entirely...

  19. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    Published
    May 24, 2018, 1:20 p.m.
    ...Arabidopsis in Education Learning Synthetic Biology with BioBuilder 3D Models for learning about CRISPR Resources...students how to perform these experiments in the beginning. In most classrooms, the laboratory experience...experiments. Notably, this plasmid harbors a multi-cloning site flanked by T7 promoters to allow for bidirectional...riboprobes) with modified nucleotides (for example, Digoxigenin-labelled Uracil) that will bind to complementary...complementary DNA (cDNA), and then use PCR and molecular cloning to insert the cDNA into the desired plasmid backbone...siRNA and are directed by complementarity to the remaining single stranded siRNA to bind natively expressed...-amputation planarians after knockdown of beta-catenin. Other gene targets do not require amputation to...

  20. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...ones? Here, I’ll cover some screening methods to identify them. Visual screening Regardless of the RNAi-trigger...organisms. When supplemented with adenine analogs such as 2-fluoroadenine (2-FA), organisms with functional...for viability (Morgens et al., 2016). Only upon combining the data generated from both methods did a more...easy creation of long hpRNA with Gateway-based cloning and straightforward positive selection of plants...assembly of intron-spliced hpRNAs through Golden Gate cloning. Artificial micro RNA and short hairpin RNA The... researcher, the traditional hpRNA and visual screening method was limited in two substantial ways: variable...morphological parameters, can be easily scored. However, determining the transcript or protein abundance of your specific...

  21. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    Published
    July 16, 2024, 1:15 p.m.
    ...pHelper plasmid containing Ad E2A, E4, and VA RNA genes; and the pRepCap plasmid containing replication factors...outcome and planning.  Both ddPCR- and qPCR-based titering assays measure physical titers, meaning the concentration...those will quantify both genome-containing (full) and non-genome-containing (empty) capsids, thus leading...TCID50 measurement reports an infectious titer, meaning the concentration of viral particles that can transduce...concentration of viral particles containing a viral genome. While ddPCR is superior in precision and variability...accessibility of instruments in standard labs. When determining titers by qPCR, we recommend using primers targeting...

  22. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    Published
    June 16, 2020, 1:25 p.m.
    ... be as pure as for other uses.   Cloning grade DNA Molecular cloning includes a set of techniques that...extraction. Figure 2: Use cloning grade DNA for direct cloning of your gene of interest into a ...in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure ...can have a plasmid or viral origin. The type of cloning approach you choose ultimately will influence the...DNA (ng range). To allow efficient and flawless cloning it is important that the preparation is pure from...the Addgene blog Browse blog posts on plasmid cloning Read our Plasmids 101 blog posts Learn about the...

  23. A Career in Grants: How To Become a Grant Professional

    Type
    Blog Post
    Published
    Aug. 20, 2015, 2:30 p.m.
    ...professionals is learning to succeed in grants through trial and error, through winning and losing funding...may consider earning a degree in Grant Writing, Management and Evaluation and/or earning the GPC credential...grant writer’, but make no mistake, researching, winning, and effectively managing grants involves much ...consulting firm, a Twitter Chat, and then our grant training and mentoring. How do I become a grant professional...to help you enter and to have a successful and meaningful career in the grants profession: 1. Build on ...There are many grant consultants who offer grant training, mentoring and coaching services, including Smartegrants...entering the Grants Profession. Whether you’re transitioning from a previous career or you’re starting your...

  24. Scientist Networking: What is an Informational Interview?

    Type
    Blog Post
    Published
    July 1, 2014, 3:42 p.m.
    ...Scientists enjoy learning new things. Building new relationships is all about learning new things from ...Training as a scientist in the academic system has many pluses. I delighted in my graduate school years...nature of academia often means that scientists-in-training rarely get the opportunity to learn about the ...science. I find it ironic that we do all of our training as scientists (5-12 years worth!) with academic... one of the best things you can do during your training is meet interesting people doing interesting things...Informational Interview it is understood to be a nonthreatening request to discuss experience, interests and...really, always). Informational Interviews are for learning and for expanding your web of connections. A mistake...

  25. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ...option to indicate separate promoters, tags, or cloning information for each insert.  Encodes gRNA/shRNA...sequence. Empty backbone A plasmid designed for cloning a gene/insert into it. Empty backbones often contain...proteins that are appended to new inserts through cloning. *Note — If you will be depositing multiple plasmids... information about the gene/insert(s). Step 3: Cloning Information Please enter the name of the vector...vector backbone information. List any promoters, cloning details for how your gene was inserted into the...for each insert individually.   Figure 9: Add cloning information for the gene or insert. Step 4: Growth...possible, we propagate plasmids in the standard cloning strain DH5α. Please indicate if your plasmid cannot...

  26. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    Published
    Aug. 19, 2014, 3:39 p.m.
    ... multiple cloning site to insert YGOI, a bacterial selection cassette for growth and cloning in bacteria...conditions. The first thing you'll need to decide when running your experiment, is which vector will best suit...bells and whistles to your construct. If you are planning to track your protein in the cell or purify it...expression vectors are inducible (e.g. pBAD LIC cloning vector (8A)) and have an epitope tag (6xHis or ...expressed protein with a specific resin (e.g. 6xHis containing pDEST-HisMBP). After purification the protein...pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check out our complete guide to empty ... Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other Plasmid Protocols List of common ...

  27. Embracing Serendipity: A Crucial Element in the PhD Journey

    Type
    Blog Post
    Published
    April 25, 2024, 1:15 p.m.
    ... explore beyond the expected. By widening your search and maintaining an openness to unexpected possibilities...simple steps to welcome these unforeseen moments. Widening your search The first step towards inviting serendipity...ultimately enhancing your project's potential. Widening your search creates fertile ground for serendipitous...could spark unforeseen connections and ideas. Maintaining openness and looseness of spirit The second crucial...element in harnessing the power of serendipity is maintaining an openness and looseness of spirit. Amidst the...of inspiration. Additionally, journaling in the morning has proven invaluable for me, allowing me to pinpoint...think beyond the confines of a predefined path, opening doors to novel ideas and approaches. Moreover, ...

  28. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    Published
    April 4, 2024, 1:15 p.m.
    ...my live-dead staining. (B) Changing the y-axis from SSC to FSC can alter the positioning and shape of ...After several steps of tissue processing prior to running my flow analysis, I can however clearly identify...’s always good to ask around for advice before running your analysis. In this case, I and other lab members...and other larger cells. Note, we don’t mind the warning message at the top, as our cells of interest do...ones (Figure 3). A more “liberal” gate in the beginning should not have a great impact on your analysis...gating pipeline, it might be worth going back and loosening the FSC/SSC gate.   Single-cell gating Once you...duplicates can’t be reliably analyzed on their staining signals. For this, you’ll need to gate based on...

  29. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...Restriction Cloning Get Tips on Analyzing DNA Sequencing Results Check out Plasmid Cloning by PCR Resources...applications range from confirming the results of cloning or knockout experiments to forensics and clinical...including easier automation, higher throughput screening, and a lower requirement for the amount of DNA...unknown sequence with restriction enzymes and then running the products on an agarose gel allows a researcher...Labs still keep restriction enzymes on hand for cloning and analytical digests. A researcher can test the...dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string of DNA (...sequencing, such as confirming the results of a cloning experiment, require only one or a few sequencing...

  30. Plasmids 101: A Brief History of Plasmids and an Improved eBook!

    Type
    Blog Post
    Published
    Oct. 29, 2015, 2:30 p.m.
    ...predictable cut site) to develop the first plasmid “cloning” experiment. Dr. Cohen and colleagues treated a...proved successful, pSC101 became the first plasmid cloning vector and molecular biology was never the same...mammalian) species were cloned into plasmids and new cloning vectors such as pBR322, pACYC, and pUC were developed...copy number vectors that could be used in these cloning experiments. Although plasmids started as a somewhat...and recombination, replication and topology, and cloning and gene expression. Just released: Addgene'...eBook include: new chapters on genome engineering, cloning, and plasmid tags, addtional content added to the... Learn more about the history of plasmids and cloning: CSHL Meeting: Plasmids: History & Biology The ...

  31. Intro to Our Summer SciComm Series!

    Type
    Blog Post
    Published
    June 9, 2022, 1:15 p.m.
    ... fall under informal learning; however, many others partner with formal learning to engage students at...environment such as the classroom or a defined learning program. Learners cannot (theoretically, at least...are interested in breeding dogs who also enjoy listening to podcasts A series of Tweets breaking down...caregiver communities  Table 1: Examples of defining your audience for science communication activities...activities  Many people are concerned that by defining a specific audience, they’ll exclude others from accessing... to you and a few other people on your team?)  Defining your audience allows you to create common ground...re ready to get into the more exciting parts - learning more about science communication itself! See you...

  32. Building and Validating Recombinant Antibodies: A Case Study

    Type
    Blog Post
    Published
    April 5, 2022, 1:20 p.m.
    ...validated. The creativity in employing a variety of cloning techniques and troubleshooting methodologies drew...colony PCR.   Fig. 1: (A) Schematic of cloning, expression and validation pipeline. (B) Schematic...the research community.    Validation For most cloning projects, the next step would be to validate your...which was observed for the hybridoma mAb.  While screening by IF-ICC worked well for identification of the...performing a BciVI digest of the PCR products prior to cloning, which in most cases left the functional light ...Approximately 70% of mouse mAbs are IgG1 subclass (Manning et al. 2012), which limits the use of subclass ...collection References Andrews NP, Boeckman JX, Manning CF, Nguyen JT, Bechtold H, Dumitras C, Gong B, ...

  33. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic ...mutations and will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR... is an alternative method that can be used for cloning or genome engineering and is based on homologous...that differ from traditional restriction enzyme cloning will be explained in greater detail. Substrate... and can be increased to as high as 25-50% by designing oligos that avoid activating the methyl -directed...

  34. Science communication: 9 Strategies to Get Your Foot in the Door

    Type
    Blog Post
    Published
    Sept. 27, 2017, 1:23 p.m.
    ...check out the distance learning courses at the National Council for the Training of Journalists (NCTJ) ...guided tours to show people where the lab magic is happening. 3) Volunteer – Step into science education Do...Learn on the job Nothing prepares you better than learning on the job. Try to get an internship at a magazine...internships to cover their annual meetings. 8) Training – Get a formal education Some people get into ...engagement for institutions and the Royal Society gives training in media skills, writing about your research and...moment, I got active with it, new doors started opening up. My current job as a Marie Curie Fellow is a... Additional Resources on Addgene.org Find Job Openings at Addgene ...

  35. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    Published
    April 6, 2017, 1:02 p.m.
    ...allencell.org and the Allen Institute website at alleninstitute.org. A classic challenge in cell biology is making...will add more to the collection later this year. Designing the donor plasmid Figure 2: Donor plasmid...make a precise cut in the genome near either the beginning or end of the gene of interest —in our case in... without outside influence (non-homologous end joining, or NHEJ), in approximately one percent of cases... process. By flooding the cell with a plasmid containing regions of high homology to the gene with the... relying instead on microhomology-mediated end-joining (MMEJ). To find out more about gene edited hiPS...allencell.org and the Allen Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic...

  36. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    Published
    July 23, 2015, 2:30 p.m.
    ...back in 2007, which function as centres for peer learning and knowledge sharing, in the form of workshops...maintains a list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One...variety of aspects from the molecular biology to learning how to grow and care for the worms. As an example...relies upon biologists and computer scientists learning and communicating with one another.      Currently...declared our makerspace an educational institution meaning that we can obtain known mutants at $7.00 a strain...optimizing the health of domesticated cats and learning about feline microbiomes. One of the aspects of...the kittybiome project, but the primary goal is learning in this case.   Funding Makerspaces One of the...

  37. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    Published
    May 22, 2018, 1:38 p.m.
    ... but educators face significant challenges in designing suitable projects. In the O'Connor lab, we recognized... about the evolution of the genes involved in methionine synthesis (5). The budding yeast, Saccharomyces...-annotated deletion strains is available (6). Methionine synthesis in S. cerevisiae (Figure 1) occurs ... transformed strain to grow in the absence of methionine (i.e. the plasmid complements the deletion). ...enable multiple student experiments We used TOPO cloning to insert the coding sequences for ten different...plasmid is controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to...strains on media that lack uracil, but contain methionine. (This allows cells carrying nonfunctional as...

  38. Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

    Type
    Blog Post
    Published
    June 12, 2014, 3:30 p.m.
    ...Addgene: Tell me about the Golden Gate Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury...already use. It makes the switch to Golden Gate Cloning easier because individual labs will not need to...make it easier for people to use the Golden Gate cloning system especially with plants in mind. When it’...There is no PCR, no gel purification; the whole cloning process is so simplistic once you have your parts... 30 constructs. They’ve never done any kind of cloning and they don’t have any prejudice about it. They... Sometimes it’s a good way to find out what’s happening in the office next to you. There is an active ... biology.  References: A Golden Gate Modular Cloning Toolbox for Plants. Carola Engler, Mark Youles,...

  39. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    Published
    Feb. 4, 2025, 2:15 p.m.
    ...requirements and expression limitations may be too constraining for some experiments, particularly in vivo ones...mice, AAV is almost entirely non-immunogenic — meaning when conducting in vivo CRISPR screens, AAV is ... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or... If you don’t feel like doubling the amount of cloning, another option is to use variants of Cas9, like...immunotherapy in glioblastoma, and generate programmable screening platforms for mouse tissues. Your AAVancement ..., Palop, J. J., & Kampmann, M. (2023). CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly...highly scalable cell type-specificin vivoscreening platform. bioRxiv (Cold Spring Harbor Laboratory). ...

  40. Transferable Skills Guide: Managing a Team

    Type
    Blog Post
    Published
    Jan. 30, 2018, 2:42 p.m.
    ...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...about management skills during your scientific training. I never thought about management skills before...grad school. Both were hard-working scientists-in-training who got the job done, but I struggled with what... he graduated. Years later I attended a formal training for new managers and I had a “Eureka” moment while... people! More specifically, they had different learning styles. One was a “Doer” and the other was a “...different levels of experience), find out what their learning styles are and capitalize on them for the benefit...it so shouldn't it be faster to do it yourself? Training certainly takes time - so plan accordingly -  ...

  41. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

    Type
    Blog Post
    Published
    Dec. 12, 2013, 3:35 p.m.
    ...that we have the beginning of control around the central dogma. We have the beginnings of computational...program created as part of President Barack Obama's Winning the Future Initiative. Endy's team made news in... - enable? Endy: The BIOFAB collection is the beginnings of what we think of as an operating system for...expression levels. The BIOFAB project is seeding the beginnings of an operating system for programming life, ...'ve described the pieces in these kits as the beginnings of an operating system or language for programming...percent of the U.S. gross domestic product – equal to mining. There is an absolutely incredible hidden story...genetic elements that allow you to begin to imagine designing 10-gene systems with quantitative precision. There...

  42. Sharing Your Lab Protocols: Using Apps to Save Time & Track Your Experiments

    Type
    Blog Post
    Published
    Sept. 23, 2014, 3:05 p.m.
    ...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the Broad Institute...this plasmid, and if it still being used. After learning of it's continued popularity, I decided this protocol...scientists, publishers, and small start-ups are turning to software apps to solve our need for quicker,...protocol listed under “Supplemental Document”: gRNA_Cloning Vector (deposited by George Church’s lab). Addgene...has also cataloged a variety of useful molecular cloning protocols, including making LB agar plates, restriction...), and now Addgene. I just began uploading our cloning protocols to the site and will keep you updated...

  43. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    Published
    July 31, 2018, 12:55 p.m.
    ...another genetic disease more prevalent or life-threatening (cystic fibrosis, hemophilia, muscular dystrophy... years ago when completing her PhD and medical training in Ophthalmology. Gene therapy was in its infancy...Importantly, it is an immune privileged compartment, meaning that it can tolerate the presence of antigens (... Congenital Amaurosis (LCA). Shortly after the cloning of the RPE65 gene, a naturally occurring animal...technology: engineering of new capsid variants, designing expression cassettes to regulate and optimize ...reports suggest that the first clinical study combining these tools may start as early as next year. Twenty... areas of science. This is hopefully only the beginning of a gene therapy revolution and the development...

  44. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    Published
    July 31, 2020, 12:30 p.m.
    ...introduced through non-homologous end joining (NHEJ). Designing an RNA editor The Zhang lab envisioned...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...gRNA spacer, as ADAR will preferentially edit an adenine if the template has a cytosine mismatch at that... natural cytidine deaminases, they evolved the adenine deaminase ADAR2DD to deaminate cytidine and used...-U edits, the authors note that it retains its adenine deaminase ability. The authors then tested the ...around the targeted nucleotide. Introduction of guanine-mismatches in the guide across from these off-target...

  45. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ...besides HR - Non-homologous end-joining (NHEJ) and alternative end joining (alt-EJ). Inhibitors to essential...harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps...’s no surprise then that these edits take more planning and finesse then their knock-out counterparts....strand break by homologous recombination.    Designing donors for HDR The basis of the HDR pathway relies...used as well. Below are some considerations for designing donor DNA for successful HDR events. CRISPR cut...reading up on validating genome edits to find screening strategies for your experiment. Also learn about...Featured Topic Page Learn about Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve...

  46. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    Published
    Jan. 16, 2020, 2:18 p.m.
    ...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...ddPCR) does not require a standard or a reference, meaning saving on reagents (and time)! AAV titering using...droplet digital PCR Droplet digital PCR involves partitioning a PCR reaction mixture into approximately 20,000...ingredients for amplification of the target DNA. This partitioning reduces the number of PCR inhibitors per reaction...replicates are built into the technology. One well (containing thousands of droplets) can be sufficient to capture...dilutions, they are transferred to a new plate containing the mastermix which includes primers and a ddPCR...small channels to create a water-in-oil emulsion containing approximately 20,000 droplets. Each droplet contains...

  47. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    Published
    Aug. 30, 2022, 1:15 p.m.
    ...You want to halt degradative processes while maintaining the structure of your samples, the relationships... it is great for preserving your samples and maintaining cellular and tissue morphology. Formaldehyde ...detergents, such as Triton X-100, Tween-20, or saponin. Each detergent has different chemical properties... and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...) Permeabilizing with the selective detergent, saponin, keeps membrane-associated proteins in place, while...fixing with formaldehyde and permeabilizing with saponin. However, if you are not worried about washing ...https://doi.org/10.1177/33.8.3894499    Im K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to...

  48. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    Published
    May 19, 2016, 2:30 p.m.
    ...Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...these plasmids, 293T cells released transgene-containing lentiviral particles into the media, which could...eliminated through the addition of a chimeric 5’ LTR containing a Tat-independent promoter. Although safer, this...both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since lentiviral...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope...lentiviral plasmid and biosafety guides before beginning your experiment. Let us know how you use lentivirus... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014...

  49. Your Top Requested Plasmid in 2015!

    Type
    Blog Post
    Published
    Dec. 30, 2015, 3:30 p.m.
    ...about pooled library screening, read our recent blog post on Genome-wide Screening Using CRISPR/Cas9. If...lentiCRISPR v2 is key to its efficient use in screening experiments; large amounts of virus are required...a single empty vector and in pooled libraries containing thousands of pre-cloned gRNAs. DNA aliquots of... If you need help cloning a gRNA into lentiCRISPR v2, you can take a look at the protocol kindly provided...Ophir, et al. "Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87... vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784. PubMed ...

  50. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...that have standard cloning sites or gateway compatible cloning sites for easy cloning of your genes of ...interneurons in mice as well as other organisms, opening the possibility of using these tools in virtually...ability to be coexcited along with EGFP while retaining an emission spectrum that is easily separable ...editing, Rees et al. created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently...reprogramming  Regulation of telomere length with TZAP Maintaining telomere length is important for cellular function...function and genomic stability, as telomere shortening leads to premature aging and cancer. The Denchi ... and repair templates of choice though modular cloning steps. The Voytas lab plans to update the toolkit...
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