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  1. Treating Muscular Dystrophy with CRISPR Gene Editing

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    Blog Post
    ...via gene therapy, so researchers have set their sights on another approach. Since shorter forms of dystrophin...to persist over time, stem cell editing would be highly desirable. Should stem cell editing occur, the ...and AAV9, respectively. Although each study used slightly different methods, they each observed the same..., the relative abundance of exon-skipped mRNA is high, likely because this mRNA is not subject to nonsense-mediated... very low to no off-target activity at the ten highest-ranking predicted off-target sites. Each paper ...CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout the body, ... rigorously examined over a long period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should...
  2. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

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    Blog Post
    ...transcribe the extremely long, highly structured HCV genome (~9.6 kb of highly structured template!). MarathonRT...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...Listen to the Hot Plasmids Podcast!  MarathonRT: A highly processive reverse transcriptase Article contributed...complex heterogeneous RNA samples. Furthermore, such high processivity enzymes and the structural understanding...antibodies: they are small (12-15 kDa), stable, highly soluble, and non-immunogenic yet are still just...
  3. Donations from Addgene to Yield Answers for Rare Disease Researchers

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    Blog Post
    ...Syndrome One of those teams is led by Claudio Aguilar (right) at Purdue University and is working to understand...forward. Xeroderma Pigmentosum Hendrik De Buhr (top right) and his colleagues at the Wistar Institute in Philadelphia...condition leaves patients incredibly sensitive to sunlight and prone to developing metastatic melanomas and... and other cancers. Joshua Wang (bottom right), also of the Wistar Institute, says that the lab got interested...Alternating Hemiplegia of Childhood Melodi McSweeney (right), a graduate student at Duke who has recently moved...returning to basal conditions normally after periods of high activity. The new work will help them to understand... Syndrome Last but not least, Meng-Shin Shiao (right) of Mahidol University’s Ramathibodi Hospital in...
  4. PiggyBac-ing Through the Genome Editing Field

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    Blog Post
    ...copies can be tightly controlled by titrating the transposase to transposon ratios. High ratios of transposase...efficiency of excision with PBx has been shown to be higher than that of the wildtype piggyBac™ transposase...importantly, unlike Cre recombinase, the PBx is highly specific, and has not been shown to cause genomic...specialized knowledge to help researchers identify the right technologies and strategies for their projects. ... 2016. PubMed PMID: 27075976. 4.  X. Li, E. R. Burnight, A. L. Cooney, N. Malani, T. Brady, J. D. Sander.... Qualls, B. M. Lewis, E. Ke, C. T. Dargitz, R. Wright, A. Khanna, F. H. Gage, and I. M. Verma, “Functional..., M. van Lohuizen, A. Berns, and D. J. Adams, “A high-throughput splinkerette-PCR method for the isolation...
  5. Viral Vectors 101: AAV Variables That Matter

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    Blog Post
    ...tricky at best, and downright infuriating at worst. While it is tempting to just dive right in and start injecting...also transduce postsynaptic cells when used at very high titers (Zingg et al., 2017), which can lead to cells... total injection volume. You’ll often hear that “high titer” is required for efficient AAV transduction...the spread of the virus.  A better way to define high titer, then, is this: It’s the optimal number of...inversely related to transduction efficacy: the higher the efficiency, the lower the titer and vice versa...needed volume (de Backer et al., 2010).  Second, high efficiency transduction coupled with a strong promoter...amount that looks effective. Ultimately, the perfect “high titer” is one that optimizes gene expression where...
  6. Scientists Map the SARS-CoV-2-Human Interaction Network

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    Blog Post
    ... in Paris and New York. We want YOU to join the fight against coronavirus We are just at the beginning...be the next scientist joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in...that we have to stick together (6 feet apart), to fight the new Coronavirus. To help get you started, here... sites - so cloning projects should be fun and straight-forward. All constructs are tagged with a 2xStrep-tag... gave a lot of background in our study - so you might want to be careful with these clones (and help us... research. For all these reasons, the QCRG is delighted to be partnering with Addgene, who will be taking...bloggers Manon Eckhardt (left) and Melanie Brewer (right) from the University of California, San Francisco...
  7. Science Career Options

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    Blog Post
    ...specific goal, and when you realise that your goal might not lead to a career you actually want, you can ...applicable to a wide range of industries and is a highly desirable quality sought after by employers. These...many employers. Increasingly, computer skills are highly desired, as roles are becoming more reliant on ...Programming skills (Python, R, SAS, HTML, Matlab etc) are highly desirable in a range of roles, including: Science...give you an idea of what additional training you might need to obtain. You'll also need to think of other...you have the relevant skills and experience.You might not normally think about it, but hobbies and volunteer... job involves traveling, showing that you are a highly organised and experienced traveler by citing specific...
  8. New and Upcoming Viral Vectors - September 2019

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    Blog Post
    ...fold increase in brightness after ~100 action potentials. This is about twice as bright as GCaMP7f and ...action potential, GCaMP7s is approximately twice as bright, relative to baseline fluorescence, as the GCaMP7f...GCaMP7f variant, and is approximately 5-fold brighter than GCaMP6s. jGCaMP7f is the fastest responding variant... variant. jGCaMP7b exhibits the brightest resting fluorescence and can be used for imaging small neuronal...processes (dendrites and axons). jGCaMP7c exhibits high contrast between peak fluorescence and resting fluorescence...lowering sensor resting fluorescence, while maintaining high peak fluorescence) and is useful for imaging signaling...
  9. Resume Writing for Non-academic Science Careers

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    Blog Post
    ...convince your potential employer that you are the right candidate for the job. How can you incorporate the...you need to have a good idea of what should be highlighted on your resume, and tailor the resume for the...programs to predict protein structures, then you might know a thing or two about computer modeling. Make...transferable skills Some of the skills you developed might not have come from the research itself, but from...universities actually have career services offices. You might think only undergraduates use these services, but... work well with your life and career plan? This might be a tough question to ask now, but the earlier ...
  10. AAV Q&A with Tim Miles

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    Blog Post
    ...standard) and reflects a desire for very high MOI on top of very high coverage. Please see this paper for ...that this will require co-delivery of two AAVs at a high MOI, often resulting in a drop in transduction coverage...particles into the same cells in vivo? A: This will be highly dependent on vector dose. The color diversity in...experiments using PHP.eB in the brain give some insight into codelivery efficiency at different doses. ...liver transduction. While we have not tried it, it might be worth investigating for both Q6 & Q7.For epithelial...that we have in house!), which seems to me a very high dosage. Any thoughts?  A: The question refers to...
  11. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

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    Blog Post
    ...and 31 times higher, respectively. In rice protoplasts, Xie et. al observed slightly higher mutation rates...this system is clearly also robust enough to ensure high levels of gRNA cleavage.   Xie et. al constructed...nucleotides to the 5’ spacers. The 3’ ends were modified slightly with either a 1-7 base poly(U)tail in tRNA-gRNA... had biallelic mutations at all five loci. This highly efficient multiplexing further illustrates the ... Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Tsai SQ, Wyvekens N, Khayter...
  12. Plasmids 101: Yeast Vectors

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    Blog Post
    ...plasmid may be expressed at higher than normal physiological levels due to high copy numbers. This creates...isolation, a well-defined genetic system, and a highly versatile DNA transformation system.Unlike most...similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle (a natural...pombe plasmids oftentimes utilize an ARS to aid in high transformation efficiency; however, this region ...antibiotic selection when working with yeast, due to high rates of spontaneously occuring resistant mutants...in the cell, thus allowing the yeast to maintain higher copy numbers. Additionally, improvements in antibiotic...
  13. A History of Genome Engineering in Popular Culture

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    Blog Post
    ...by large DNA recognition sites, which makes them highly specific.  Jurassic Park by Michael Crichton was...Jurassic Park (1997, 2001, 2015, and 2018) were highly successful in the box office, indicating the insatiable...increase specificity. This modular approach made ZFNs highly appealing to researchers.  TALENs In 2010, transcription...travels. The movie sees the USS Enterprise crew fight against a genetically engineered superhuman Khan...cellular DNA repair machinery, which can lead to high frequency of indel formation in the genome and are...involves The Rock and his super giant albino gorilla fighting off a monstrous wolf and crocodile, that were ...Editing Trading Card seen in this blog post?Download high resolution versions here.  Many thanks to our guest...
  14. Transferable Skills Guide: Teamwork

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    Blog Post
    ...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today: Teamwork. My first experience...was I to dispute history? And boy did I make the right decision. If I’d spent my senior year isolating ...colleagues, I can also orchestrate the formation of high-functioning-but-temporary teams that have a specific...providing technical customer service. Team members are highly trained, and though there are necessary redundancies...skills - you just want the necessary skills. You might find that your next paper is going to require help... of more than one person. A selfish part of you might not want another person’s name on your next paper...
  15. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ...clone out of 104 to 105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability... center of the sequence. Since lambda red has a higher recombination frequency when the lagging strand...strands. One of the two oligos will recombine with a higher efficiency than the other and you’ll find out empirically...frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing oligos that avoid activating...deletion of >20 nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase to generate insert ssDNA...these strains, expression of exo, beta and gam is tightly regulated by the endogenous phage promoter pL and... allows for a mobile recombineering system, but tight regulation of expression is required for a successful...
  16. Live and Let Dye: Self-Labeling Protein Tags

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    Blog Post
    ... (TMP-tag) (Miller et al., 2005). Although this tight interaction is not covalent, later work showed that...systems can be complicated to implement, they can be highly specific with a small and minimally-disruptive ...  Applications Organic dyes are typically both brighter and more photostable than fluorescent proteins...in a new fluorescent ligand and see the results right away. Or perform pulse-chase experiments by sequentially...membrane. So, you should investigate your options and weigh the potential benefits and costs before investing...investing in one approach or the other. Finding the right tag for you Protein-based tags typically provide ...the fluorophore’s microenvironment to enhance brightness, stability, or other properties is an active ...
  17. Antibodies 101: Validation

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    Blog Post
    ... antibody has arrived, hooray! Before you jump straight into the exciting new experiments that you’ve ... their ability to bind to molecular targets with high affinity and specificity. However, there are countless...validation in different applications. Here, I want to highlight the general suggestions given by the International...your bioinformatics skills (or your friendly neighborhood bioinformatician) to look at RNA expression ... tissues.  From this data, you see that there is high RNA expression in the retina and in parts of the...binding to something unexpected. This approach has high specificity, but you need to be aware of any possible...for immunocapture applications and is amenable to high-throughput analyses, however, it requires access...
  18. A Guide to Getting Started in Undergrad Research

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    Blog Post
    ...Nye in their lab coats handling glassware with brightly colored hazardous liquids. Working in a lab is...school, med school, industry) Other roles you might see Lab manager: managerial duties ex: order materials...makes them excited to do what they do. I would also highly recommend talking to other undergraduate researchers...choosing a good scientific mentor and choosing the right lab. Remember that your time is valuable! Don’t ...scientists are all just people. I’ve found that in the right environment and with supportive mentors, being open...was a bit of a foreign concept to me when I was in high school, but I’ve learned the value in finding mentors...broadening your experiences! These applications can be highly competitive, so asking for help from mentors and...
  19. The time and cost required to make a plasmid

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    Blog Post
    ...significantly reduce costs of producing plasmids with high-throughput methods that save on reagents, consumables...many labs are quite happy to share, labs that get high volumes of plasmid requests can end up refusing ...traditional PCR/digest/ligate methods and picking the right one can save you time if you’re working at speed... that works for your experiment. Make your work “high-throughput” Instead of making one plasmid at a time...t scale or have limited scaling. The downside of high-throughput work is that you give up a lot of flexibility...minute experiments or changes. Fun fact: Addgene’s high-throughput approach means we virtually always use... Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific ...
  20. CRISPR 101: Cas9 vs. The Other Cas(s)

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    Blog Post
    ...abilities of the lesser-known Cas enzymes while also highlighting their similarities and differences to Cas9. ...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets ssDNA...ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a single mismatch in the...sequence. Why would you want to give it a go? The high specificity of Cas14 makes it a promising detection... Fast facts – PAM requirement: none. Best for: high fidelity ssDNA targeting. RNA targeting Cas enzymes...enzymes are worth taking a look at. Choosing the right Cas can really put your experiments Nsync.   References...
  21. Antibodies 101: Polyclonal Antibodies

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    Blog Post
    ...conformational state may change. If your protein might become slightly denatured during your experimental protocol... or a goat) is injected with an immunogen. This might be the full-length protein that you want to generate...that recognize your target protein without causing high background due to cross-reactivity. When to use ... in many labs. Polyclonal antibodies also have a high sensitivity to the target protein due to their ability...or recombinant antibody instead. Otherwise, you might experience variability in the strength of staining...strength. In general, polyclonal antibodies produce a higher level of background noise compared to monoclonal...
  22. 5 Steps to Writing Grant Proposals for Grad Students

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    Blog Post
    ...given problem, Empowers the writer, And offers insight into a career as a Professor. Step 1: Your ...time to review the literature and offer educated insight into what the desirable features of future technologies...field. Research strategy The Research Strategy highlights i) the significance and innovation of the project...statements. While some of the statements may be slightly far reaching, remember that you are writing a ...text. Specific aims The Specific Aims section highlights each milestone that must be met in order to deem...
  23. How to Prepare for an Industry Interview

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    Blog Post
    ...about your prior research. This is a chance to highlight your prior accomplishments and demonstrate your...skills that make you qualified for the position. Highlight the outcomes or deliverables of your work, such...extraversion for me. Make sure to get enough sleep the night before. Have your morning coffee, if that’s part...part of your daily routine. Stay hydrated. You might have lunch on your schedule, but it will probably be...’t have technical challenges, your interviewers might.  You’ll probably still be required to give a job...
  24. 6 Tips for Grant Writing

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    Blog Post
    ...post was contributed by guest blogger Sean Mac Fhearraigh, co-founder of www.assaygenie.com. No matter ...required and no one lab or professor becomes an overnight success. Below I have detailed some tips to improve...in different fields that can provide different insight into an area - not just working within the established...and possibly a new perspective on the field. 3. Highlight your network and your collaborators It’s a good...collaborators can also help you get some critical insight into what you're doing wrong. All this helps to...  Many thanks to our Guest Blogger, Sean Mac Fhearraigh! Seán was a PhD student at University College...
  25. Plasmid-based Recombinant Monoclonal Antibodies: What They Are and Why You Should Be Excited About Them

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    Blog Post
    ...Left) The light chain and heavy chain genes of the antibody are encoded in a plasmid. (Right) An assembled...If you were born before 1985, you might remember going to the store and buying CDs when you wanted to...cell line. The genes encode the heavy chain and light chain for the antibody and when translated into ... reagents. These include conventional heavy and light chain recombinant monoclonal antibodies as well ...encoding antibodies, scientists will be able to make higher affinity antibodies, modify antibody function, ...10.1080/19420862.2018.1445456 Crosnier C, Staudt N, Wright GJ (2010) A rapid and scalable method for selecting...
  26. Hot Plasmids and Viral Preps - March 2021

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    Blog Post
    ...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...be easily visualized.  The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...Rex's lab.  vfCRISPR, or very fast CRISPR, is a light-inducible CRISPR system developed by Taekjip Ha’...target DNA without cleavage until it's activated by light. Once activated, Cas9 cleavage can induce DSBs within...
  27. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

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    ...Although other methods have been able to achieve much higher activation, dCas9-VP64 is great for experiments...targeting single genes, SAM consistently shows the highest levels of gene activation compared to other CRISPR...Performance Generally, VPR is found to have significantly higher activation levels than the initial dCas9-VP64 activator...rearranging chromatin structure. This method has shown higher activation levels than SAM in some loci across ...has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou et al., 2018). ..., Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming...https://doi.org/10.1038/nmeth.3871 Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...
  28. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

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    Blog Post
    ...current findings in nucleic acid science. I was delighted to see that the majority of attendees were scientists... enjoys preferential governmental policies for a high-tech development zone so many science and technology...fauna. I tried to find out what certain ingredients might be used for (like what would one take dried seahorse...blot results and we didn’t give tRNAs much thought. High throughput transcriptome sequencing methods including...families and provide a way to guide inheritance that might be less error prone than DNA replication. He too...things.” He further cautioned the distinction of “higher” and “lower” life forms as we shared millions of...the bacteria sensitive to known antibotics. These might be used on hospital surfaces and in hand sanitizers...
  29. Plasmids 101: Modular Cloning Applications and Kits

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    ...basics of the technology. In this blog, we will highlight some common MoClo applications and kits. Keep ...beyond what we discuss. If you can think it, MoClo might help you achieve it! MoClo applications and specialized...cassettes (Shaw et al., 2023). Those looking to express high levels of their gene of interest in yeast should...10.1021/acssynbio.6b00337. Lee, Michael E., et al. “A Highly Characterized Yeast Toolkit for Modular, Multipart...s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex Editing: One‐shot Generation...doi.org/10.1111/tpj.15197. Grützner, Ramona, et al. “High-Efficiency Genome Editing in Plants Mediated by ... Addgene blog Plasmids 101: Golden Gate Cloning High Complexity Golden Gate Cloning with NEB’s DAD New...
  30. Quick Guide to Working with Drosophila Part 1: Getting Started with Flies

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    Blog Post
    ...daytime. They mostly stop emerging at night. An incubator with a light cycle makes for very predictable work...temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies develop faster but...When flies first emerge, their bodies will be very light in coloration and can look very plump. On the side...flies. Like Addgene, the Drosophila community is highly sharing-oriented, and there are many resources ...your fly gene using the search box in the upper right hand corner. They have video tutorials under the...useful information on topics such as choosing the right balancer chromosomes to use. If you’re not ready...
  31. xCas9: Engineering a CRISPR Variant with PAM Flexibility

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    ...should be easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting...near your insertion site, the corresponding gRNA might be undesirable due to potential off-target sites...they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9: NGT (4.5-fold efficiency...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly,...generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing efficiency over that ...Evolved Cas9 variants with broad PAM compatibility and high DNA specificity." Nature (2018) doi: 10.1038/nature26155...
  32. Antibodies 101: Conventional vs Spectral Flow Cytometry

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    Blog Post
    ...flow cytometry During conventional flow cytometry, light emitted by a fluorophore is guided to a single detector...on the other hand, utilizes the full spectrum of light to distinguish one fluorophore from another. Instead...upwards of 30-40 colors), spectral flow cytometry might be the better choice. Of note, there is no minimum...of the markers you are including in your panel. Highly expressed markers should be paired with dimmer ...markers with low expression should be paired with brighter dyes, so they can more easily be detected. Cytek...cells when designing your panel. If you have two highly overlapping cellular markers, it is important to...on the Addgene blog Antibodies 101:Selecting the Right AntibodyAntibodies 101: Secondary AntibodiesAntibodies...
  33. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

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    ...False, width='600', height='189', player_id='31668202421', style='text-align: right;' %} Figure 3. An...data with a 7-day rolling mean gave us a crude insight into the magnitude of the effect COVID-19 had on...there are many new COVID-19 cases being reported (right, red), likely due to quarantining {% video_player...most impacted or least impacted. These results highlight the instability and uncertainty in estimating ...methods for fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate...
  34. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

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    ...outperformed another CRISPR library with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries...CRISPRi sgRNA library While CRISPRko libraries are highly effective at identifying strong hits in genetic...because it's essential for viability. In addition, high copy number genes may mimic viability genes as they...knock-out library while mitigating toxicity induced by high copy number dsDNA cutting. A Dolcetto library containing...wide CRISPRko, CRISPRi, and CRISPRa libraries are highly effective for probing gene function while using... 1. Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation...
  35. SciComm: Taking Science to Elementary and Middle Schools

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    Blog Post
    ...a new coordinator for the annual Math & Science Night, I took the reigns. I also volunteered when our ... started coordinating the annual Math & Science Night, a chance for parents and other members of the community...activities. If your school doesn’t have a Math & Science Night (or some equivalent), here are some quick tips to...activities we had for my school’s Math & Science Night. There are plenty of places to get ideas for science...for all of these questions, and for any topic you might present to middle schoolers, is to provide current...again next year. There are many reasons why you might want to reach out to your school community as a ... Elementary for trusting me with Math & Science Night, and a special thanks to Joanne Marks, Jonathan ...
  36. Negotiating Work and Life: How to Find the Joy

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    Blog Post
    ...Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work/life balance. Is it truly possible to...have one-half of the guilt. You can handle that, right? Big tip: this is an important topic to discuss .... The same with sheets – wash them and put them right back on the bed to save a step! There is more than...situation. Go to church, join a club, meet the neighbors and create a network of support to help with elder... elder care, carpooling, cooking on nights when you can’t get home, babysitting or running errands. Give...very, very fast! I will never forget the time my eight-year-old daughter vehemently reminded her doctor...wait and some things can’t. I kiss my kids good night every evening I am home no matter how busy I am....
  37. Transferable Skills Guide: Career Planning Resources

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    Blog Post
    ...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today: Career Planning Resources...graduate school and beyond is to live in your lab, right? Wrong! While it is clearly a good idea to be diligent...there are different types of conferences that you might attend. Societies, such as the American Society ...contact an email with the relevant info and they just might personally hand your credentials to HR, greatly ... institution’s career center. Alternatively, it might even be a colleague. A mentor does not necessarily...
  38. Hot Plasmids - November 2023

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    Blog Post
    ...such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing efficiencies...Every few months, we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...the utility of this technique.  By engineering a light-sensitive LOV domain into the TurboID enzyme, Lee...LOV-Turbo becomes active when illuminated with blue light, catalyzing the biotinylation of nearby proteins...Cheah, J.S., et al. (2023). Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal...
  39. Antibodies 101: Immunoprecipitation

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    Blog Post
    ...from magnetic beads are equivalent, if not slightly higher, than those of agarose beads, despite the size... method is appropriate for most situations.  A slightly different method is desirable if the antibody ... crystallizable region (Fc) of antibodies with a high affinity (Hjelm, 1972, Bjorck 1984). Protein A, ...beads, break weak protein interactions, and limit high throughput procedures. However, agarose beads are...the protein of interest. The wide availability and high specificity of anti-epitope antibodies make this...Tagged proteins are typically expressed at much higher levels than endogenous proteins. Data obtained ...
  40. Important Considerations in Optogenetics Behavioral Experiments

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    Blog Post
    ...Central PMCID: PMC3150193. 8.Han, Xue, et al. "A high-light sensitivity optical neural silencer: development...in your brain region of interest could be low or highly variable without the behavioral test. Another readout...important in memory, an appropriate behavioral test might be contextual fear conditioning (reviewed in [4]...optogenetics setup (viral expression, efficiency of laser light transmission to the target neurons, appropriate ...mistakes. The easiest way to learn optogenetics is straight through the source - find a lab within your own...Filippo, and Claire Wyart. "Optogenetics: a new enlightenment age for zebrafish neurobiology." Developmental...
  41. Plasmids 101: E. coli Strains for Protein Expression

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    Blog Post
    ...need an expression strain? Protein expression from high-copy number plasmids and powerful promoters will...typically expressed in E. coli engineered to accomodate high protein loads using inducible promoter systems (...to all or most expression strains to accommodate high protein levels including:  ompT: Strains harboring... (DE3) pLysE* Chloramphenicol (pLysE) pLysE has higher T7 lysozyme expression than pLysS; expression vector... (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin reductase and glutathione reductase...—the difference being that the latter provides tighter control of basal expression. What if I don't see...media such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media...
  42. What's New in CRISPR - Winter 2018

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    ... In this quarterly blog series, we’ll highlight a few of the new CRISPR plasmids available at Addgene...recently developed a platform called SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) to detect... this further to provide multiplexed detection, higher sensitivity, and more. Read the paper in Science...developed dual-nuclease Cas9-Cas9 chimeras with higher target site activity and more predictable and precise...inhibitor, with the LOV2 photosensor from Avena sativa, light-mediated genome editing is now possible. Learn ...
  43. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

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    Blog Post
    ...canonical base editing window, base editing has higher efficiency and fewer indels than prime editing....However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning...varies widely — it still almost always results in higher ratios of desired edits to indel byproducts than...average 2.3- to 5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1 ...process of double nicking increases indel formation slightly. Designing the sgRNA with a spacer that only binds...asked. Hopefully this table helps you keep them straight! Innovation Purpose PE2 PE3 PE4 ...PE6 PE7 Pentamutant reverse transcriptase Higher efficiency ✓ ✓ ✓ ✓ ✓   ✓ Second...
  44. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

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    Blog Post
    ...institutions of higher education nonprofit entities related to or affiliated with institutions of higher education...reserved for individuals with a US Master’s degree or higher degree. These numerically limited visas are referred...theoretical and practical application of a body of highly specialized knowledge in a field of human endeavor...the cap if the applicant works at institutions of higher education, a related or affiliated nonprofit entity...interest waiver criteria however the EB-1A requires a higher standard of proving that you are one of the top...showcases (not usually applicable to scientists). High Salary –not relevant to most postdocs. Commercial...circulation) in the field EB-1Bs historically have a high rate of approvals with USCIS due to the fact that...
  45. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

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    Blog Post
    ...Proximity labelling relies on the labelling of neighboring proteins within a cell with biotin. Biotin, is...avidin coated purification techniques. Due to the high affinity of biotin for avidin, proximity labelling...promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin based proximity labelling...from the ligase and bind to exposed lysines on neighboring proteins. Thus to capture protein complexes, ...electron-rich amino acids such as tyrosine on neighboring proteins resulting in their biotinylation. The...The advantage of APEX is its speed, labeling neighboring proteins in minutes rather than hours like BioID...sensitivity. Sensitivity could be increased with higher expression levels but for many fusion proteins ...
  46. Bringing Sustainable Practices to the Lab: Innovations

    Type
    Blog Post
    ...some of these plastics are high grade and can be washed and reused. This might sound risky in terms of contamination... in a high-throughput and efficient way? Grenova, for example, offers automated, benchtop, high throughput...are an accepted part of lab culture, but with the right incentives (for example, by grant agencies), we ...behavior and innovate solutions and practices to meet higher environmental standards. Find some easy action ...
  47. Plasmids 101: Walkthrough of Addgene’s Snapgene-Powered Quality Control Process

    Type
    Blog Post
    ...contains a region with a high amount of guanine and cytosine nucleotides, we might need to send it for Sanger...to confirm the sequence of a plasmid and we’ll highlight some of the new features available on our website...our first clue about where the gene of interest might be located. By easily using BLAST to check the translated...repetitive region is present in the plasmid, we might need to perform a restriction digest to confirm ...
  48. Oh, The Places You Can Go: Careers in Science Communication - Product Development

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    Blog Post
    ...get to where I was. It was really luck of right place, right time, and having a good interview experience...educators, mostly elementary school, middle school, and high school.  I’m the only science person of the bunch...plans was paved with lots of writing, hard work, a slight detour, and a bit of luck. This journey started... editing These teaching experiences definitely weighed in Caitlin’s post-grad plans, but in the end, writing...services. “I was working during the day and editing at night. It was not the easiest 6 months. But it gave me...
  49. Running for Rare Disease, Running for FOP, Running for AJ

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    Blog Post
    ...raging night, With strong heats full, our souls ignite, When all seems lost in the War of Light, Look ...After three kids, I had put on typical middle age weight and “exercise” consisted of taking walks in the...career as Senior Director of Biology, is helping us fight against FOP in two ways. First, Addgene is distributing... their plasmids with Addgene and join us in the fight against FOP. Second, Addgene will be hosting the...                                    -for HOPE burns bright!"   Many thanks to our guest blogger Kurt Swanson...
  50. Five Questions to Ask If You're Deciding Between a Master's or PhD Program

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    Blog Post
    ...Scientist I/II. Promotions don’t happen overnight though, and it might take longer to advance without a PhD... few questions to help you decide if a master’s might be for you. 1. Are you interested in master’s-...with a PhD, a master’s is less specialized so it might lend itself more readily to a switch. 4. Do you ...the bench. So if you enjoy wielding a pipette, it might make sense to earn a master’s instead of a PhD. ...good fit with your interests and career goals. You might still decide to earn a PhD, but if I had to do grad...
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