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... focus on how to choose an advisor/principal investigator (PI) that will also serve as a good mentor. ...advisor is a good mentor? You have to ask a lot of questions. Personality and style conflicts are a common ... determining a good fit. Here are just a few suggestions for determining whether an advisor will be a ...potential advisor and don’t be shy about asking hard questions. Talk to as many lab members as possible. Try ...Ask alumni of the lab. Most labs have websites listing alumi and it is easy to use publication records...this blog post. This document includes a list of questions to ask your potential advisors and other related...Check Out Angela Depace's Talk on "Precision and Plasticity in Mentoring" Don’t ignore warning signs If lab...

...overcome. Conflict, and its inherent tension, is interesting. It’s what drives a story forward - and this ... that the superheroes win in the end. But we’re still invested in seeing how they do it.    Finding...your formal scientific communications, they are fantastic fodder for science communication. The trick is... the pieces that make you think, “Oh, that’s interesting! How did you solve that problem?” Those are the...big-picture problem, by asking your audience a simple question, like “Have you ever thought about how X works...conflict, you’re encouraging your audience to ask questions. Why didn’t the experiment work? What did those...preventing them from moving forward? Answering these questions will bring you and your audience - via the scientific...

...What tools are still missing from the optogenetics toolbox? Boyden: That's a good question. We've made a...about this latest optogenetics advance, the big questions that motivate his interest in neuroengineering...neuroengineering, and some of the optogenetic tools that are still missing. Optogenetic tools for neuroengineering Addgene...compete against each other. Getting two-color stimulation has been a holy grail of the field. The problem...fundamental understanding of the brain, which has humanistic and philosophical implications. As an example... a lot of progress. I would say we still would like to find ways to make these tools more selective for...calcium or some other specific ion that would be interesting. It would also be great if you could make neural...

... so minimal experimental testing is required. So how can you do this? Testing all IFNs is completely unnecessary...are a matter of sensitivity. Therefore, the key question is not whether off-target effects exist, but what...simple: choose the least active SpCas9 variant that still efficiently cuts your on-target site. If a nuclease...). However, only the least active variant which still retains on-target activity (highlighted by a bold...higher-activity nucleases, the weakest variant that is still active can achieve off-target–free editing for this...Figure 2). Identify the least active variant that still shows the right amount of on-target editing. This.... (In the example in Figure 3, the least active still working variant was B-HypaR, thus variants with ...

...et al. "The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations....Stochastic multicolor labeling is a popular technique in neuroscience and developmental biology. This... persistently fluorescent during development. Stochastic multicolor labeling systems, many based on Brainbow...for improving the level of detail possible for stochastic multicolor labeling of the brain. Labeling Drosophila... Cre recombinase can be used alongside common, existing tools for fly research that use Flp recombination... to describe the self-renewal of cells in the intestinal crypt, and since then has become a powerful tool... researchers who developed Tetbow. Conclusion Stochastic multicolor cell labeling systems allow single...

...Harvard Business Review article on procrastination recommends testing out how much time you can possibly...conceptual chunks: prioritization, planning, and procrastination. Time management through prioritiziation Every...option. Even if the full experiment fails, you’ll still have made small accomplishments each day that should...Eric before doing anything else. Preventing procrastination Even if you’ve done all the prioritizing and...and planning in the world, procrastination can easily prevent you from making progress on a task. For ...few things I’ve found useful to avoid such procrastination: Remove distractions: Probably the easiest...book, “Bored and Brilliant” for learning more fantastic reasons to remove yourself from distractions every...

...being able to ask follow up questions, or ask people to rephrase questions was key. I had also hoped that...that’s what keeps things interesting. And hopefully they’ll be just as interesting as the finished product...familiar with this, it’s a method for building and testing new products or methods, using metrics and feedback...webcams.  Build: I sent the participants interview questions they could answer, and I’d cut together a video...the office had partially reopened. But there were still many restrictions in place, most importantly, masks...would allow me to set up a camera in a room, but still have control over focus, zoom, stop/start record... to mount a battery to it if necessary. I could still control the camera from a distance if I needed to...

...listeners to ask questions and record those that were asked. Reflect on the questions and come up with...Google.slides or Powerpoint file right now to capture interesting key results, striking images, creative ideas,... write itself when the time comes. 5. Choose interesting content Talk about something you know a lot about...commercialized, just talking about the possibility is interesting. Check out Joanne's Reddit AMA 6. Practice, practice...everybody loves a great animation. 8. Predict questions, prepare answers Speaking pros always have a few...always better than too long, and ensures time for questions. Don’t be tempted to cover too many things. One...this enough…more is almost never better. 10. Be enthusiastic You don’t have to emote beyond your comfort ...

... to the products of parental plasmid digestion (blue). Digestion with enzyme “B” detects primer multimerization.... Many thanks to our Guest Blogger Kristian Laursen! Kristian Laursen is a researcher at Cornell University...This post was contributed by guest blogger, Kristian Laursen from Cornell University. Site directed mutagenesis...can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner. The approach...perform alanine scanning mutagenesis or targeted substitution of key residues), and in SNP analysis (to introduce..., the template is removed by DpnI restriction digestion leaving behind only the mutagenized plasmid. Although... bases at the 3’-end interfere with plasmid reconstitution, and consequently Taq polymerases cannot be...

... µL size serotype testing) pAAV-CAG-GFP 50465 AAV8, 9 (20 µL size serotype testing) AAV-hSyn-EGFP ...size serotype testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP...Our new AAVs include: Control AAVs for serotype testing jGCaMP7c for calcium detection PSAM4 GlyR for chemogenetic...control AAV that scientists can use for serotype testing. These samples are available in various serotypes...encoding GFP. Check out our inventory of serotype testing AAV New control AAVs Plasmid Serotype Name ...This variant exhibits high contrast between its resting state and its calcium-bound state, which was achieved...next 6 months, pending quality control. For an estimate on availability date or to be notified when a ...

...real-time monitoring of signaling pathways, investigating RNA regulation, tracking protein degradation...

...Associate Director of the Cancer Program at the Broad Institute of Harvard and MIT. The notion of cancer precision... many tricks up their sleeves and most patients still don’t benefit from this approach. One central bottleneck...We've developed a new plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we...have been two huge challenges preventing us from testing this hypothesis: There was no large scale, broadly...cellular pathways). It wasn’t clear whether gene-agnostic bioassays could accurately test for molecular ...variant function and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome...this first challenge, we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely...

...David Root from the Broad Institute and Alex Chavez from the Wyss Institute were instrumental in helping...School, and Jonathan Ting’s lab from the Allen Institute have supported the AAV initiative from the beginning...at Addgene. Our Board helps keep our focus on assisting scientists and accelerating research and discovery...Team has been developing expertise in answering questions on virus and helping provide educational resources...word out about this new resource and providing fantastically useful educational content on viral vectors....internationally and be ready to provide extensive logistics support for export and shipping on dry ice. The...permission from relevant plasmid depositors and their institutions to distribute viral preps derived from plasmids...

...lead to cystic fibrosis, one of the most prevalent inherited diseases. The authors investigated the role...One common strategy is protein overexpression — boosting levels so interactions aren't missed. But this...knock-ins maintain the native protein context, but they still depend on antibody-based detection or western blotting...binding to its complementary partner LgBiT, reconstitutes a fully functional luciferase enzyme (Figure...HiBiT provides a means to quickly answer research questions before creating knock-ins.  Figure 1: ...these variants of p53 shape chemosensitivity and mechanistic drug responses (Kamio et al., 2025). They designed... to characterize the proteasomal degradation of cystic fibrosis transmembrane conductance regulators (...

...on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and the Joung Lab similarly ..., researchers from the Zhang lab at the Broad Institute, the Joung lab at Harvard Medical School, and ...activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure of the Cas9... Slaymaker et al. made a variety of alanine substitutions throughout the groove in 32 separate Cas9 mutants...multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A) and eSpCas9...interaction sbetween the non-target strand and Cas9, Kleinstiver et al disrupted interactions between Cas9 and...We hope one of you will be able to answer this question very soon! We look forward to seeing how the research...

...the first time, have lots of questions. To keep up with all these questions, Dr. Le Cong and Dr. Feng ...CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR", short-hand for ...developed a Google Forum to allow scientists to ask questions and get answers on the fly. But just like the ..., to organize the CRISPR forum's most popular questions and answers. Designing your CRISPR genome editing...expression, though it is possible for the plasmid to still express without the G. Because the G is only one...the efficiency will be lower, although this might still be okay if you are introducing a selection marker...insert (gRNA) in pX330/pX335 with BbsI/AgeI double digestion. As a successful insertion will destroy the BbsI...

...list of Fragmid destination vectors currently available. Table 1: Fragmid destination vectors Organism...depicted. (Bottom) Schematic depicting available destination vectors for various delivery methods, along with... with compatible inserts. All destination vectors contain an ampicillin resistance cassette and an origin...components you want, simply assemble them into a destination vector via Golden Gate cloning, perform an exonuclease...sequence for every individual assembly. What destination vectors are available? What you’re building ...Organism Delivery Type Destination Vector Mammalian Lentivirus pRDA_512 (RSV-driven; lentiGuide-based...Mammalian Transposon pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid pRDA_791...

...students and postdocs broadly – and thus PI’s and institutions in turn. The first is the passing and upcoming...the NIH recommended pay scale – used by many institutions to set postdoc wages. The second event of note...students will pursue this newly available avenue. Interestingly, both of these changes share a common thread...simply being able to categorize all postdocs at institutions, will be challenging. At the same time, there...level, FoR is currently compiling a database of institutional responses to the FLSA ruling. Similarly, from...move in the right direction, the increase notably still does not meet the suggested postdoc salary ($50,...advocating for change: from developing a strategy to distilling a message for the appropriate audience and communicating...

...types of difficult people: Hostile-aggressives, who are usually antagonistic and impolite, Complainers...work with difficult people? Each time I ask this question at my workshops, I get nods from nearly every ...unresponsive clams, who seem to ignore you and your questions, Super-agreeable friendly types, who don’t follow...have time to mentor you, and Micromanagers, who question every detail of your project and work hours. ...example, you might misunderstand your supervisor’s suggestions and take your project in the wrong direction....chance to review what you have discussed and add suggestions if needed. 7) Always follow through on your end...is a difficult person, the above strategies will still be helpful but you will need to be more assertive...

...cells. These properties make lentiviral vectors fantastic options for delivering shRNA, CRISPR/Cas9 components...membrane pores) transfection. These techniques are still used and efficiently transfer plasmid DNA efficiently...way to re-create a functional virus. Indeed, by testing several vectors and packaging cell line combinations...vector. Lentiviral vectors Although retroviruses are still in use, considerable efforts have been devoted to...However, in this vector production system, there is still a potential risk for the generation of replication...however improving the safety of these vectors is still an active area of research due to the possibility...Replication-incompetent lentiviruses with human tropism can still infect the researcher and insert into her/his genome...

...KRAS mutations: diagnostic and clinical implications." The Journal of Molecular Diagnostics 12.4 (2010): ...Hendel et al. 2015). Low detection rates mean the investigator can be relatively certain that their samples...off-target mutations are essential data points for investigators looking to definitively link genotype and phenotype...algorithms, their genome-wide search criteria are not exhaustive. Mismatch tolerance settings are often limited...larger mismatches (e.g. six nucleotides) that may still lead to off-target double-stranded breaks (Tsai ...Programmable, can detect DSBs in live cells Not as senstive as other unbiased methods, high background BLESS... multiplexing) Requires large cell population, senstive to time since cell fixing HTGTS Not reported...

...your poster visitors had a great idea or interesting questions worth discussing with your lab. If you made...conference Take notes during talks. If you have questions during the talk, jot them down for the Q&A immediately... went to, I made it a personal goal to ask one question each day (this may be more or less difficult depending...speaker from the conference if you have additional questions. If you or your lab identifies a potential collaborator... want to look through themselves and then ask questions. Practice your presentation for both instances...and say something like “Let me know if you have questions or if you want me to walk you through the poster...a notebook handy so you can jot down notes or questions from your poster viewers and trade contact information...

...healthy and diseased states can also provide interesting insights into disease mechanisms and protein ...interest from its production site to its final destination. As an example, most secreted proteins are produced...pathway to their subcellular or extracellular destination. At the end of their life cycle, proteins may...plasmid collection from Sue Jaspersen (Stowers Institute) and Mark Prescott (Monash University). You can...subcellular structure of interest in the Allen Institute for Cell Science Plasmid Collection. You can also...analysis software produces quantifiable results and statistics that can be used to compare between different...direct molecular interaction without further investigation. One fluorescent microscopy method commonly ...

...can answer some of the surprisingly technical questions elementary students can ask. It doesn’t just need...some accountants running our math tables!  Be realistic about what you can teach  5-10 year olds in a ...some crowdsourcing on Twitter and got lots of suggestions. Here are just a few resources: Steam Powered...for 8th graders was a little intimidating. Too simplistic and I would insult the students. Too complicated...  The overarching guideline for all of these questions, and for any topic you might present to middle...talking to 8th graders was the broad range of questions I received. I gave my presentation five times ... it, and trying to answer some of their great questions, was immensely gratifying. I came out of the experience...

...Planning Tips! 1. Take suggestions Some of Addgene’s best events have been at the suggestion of an employee,...imagine, it keeps all of us quite busy. Though there still isn't time for goofing off, the company culture ...Candidates who interview at Addgene frequently ask the question, “What’s your favorite part about working at Addgene... Addgene?” Most Addgenies reply with an enthusiastic, “Our company culture! Addgene is the best place ...animation, but the gist is a common answer to this question.  We truly work in a unique environment where,... familial aspect of Addgene. As our size has drastically increased, it would have been a tight squeeze... it was just beginner’s luck, but I’m feeling optimistic for the next tournament). Minus a little rain...

...no surprise that the U.S. Bureau of Labor and Statistics shows that employment of grant writers is expected...professional. Dr. Cook served as a principal investigator for several research grants. One of the benefits...benefits of being a principal investigator is that you are asked to review grant proposals from others. ...career in the grants profession: 1. Build on your existing skills & passion  Skills such as research, collaboration... development experience and to build upon your existing skills.  2. Find your niche Identify and develop...career goals, identify your niche, and answer questions when you’re negotiating a new job or a new contract...credential from the Grant Professional Certification Institute (GPCI). GPCI evaluates a candidate's expertise...

...Frequently asked questions Now let's get into some of the nitty gritty and answer the questions those of us...reagents as outlined by your institution or country. Have any other questions? Feel free to post them in..., and more. Not surprisingly, we receive many questions from scientists all over the world looking for...answers to our most frequently asked lentiviral questions. This useful resource from Addgene Depositor ...quickly check that a construct is functional before investing time and resources into making a stable cell line...is useful in gene therapy applications where a distinct subset of the cellular population needs to be ...aspects of lentiviral transduction along with suggestions on how to lower the risk. Biosafety should always...

...yet powerful way to tag cellular proteins and investigate protein localization, interaction, and expression...reversible photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation...victoria) GFP (wtGFP) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population...can occur multiple times by activating with two distinct wavelengths of light (see Table 2). The best known...usually preferred for studying single protein characteristics, as the monomeric state is less likely to interfere...super-resolution imaging usually have the following characteristics-high contrast ratios, bright fluorescence, ...conventional FPs for super-resolution imaging, they still do not emit the large number of photons achievable...

...2):a001107. PMC PMID: 20182618 . Do you have suggestions for other plasmids that should be added to this...

...last reviewed: 10 September 2025 Do you have suggestions for other plasmids that should be added to this...

...to the other six mentoring questions in future posts. If you have a question about mentoring that you want...Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this blog... series, I plan to provide answers to the top questions scientists ask me about mentoring. Here they are...’ – inspire you.” “Also a mentor can give you realistic expectations. (Don't think you can finish that... is a sounding board--a good mentor asks more questions than answers them.” A good mentor does all of...lighthearted, the article does highlight key characteristics of a good mentor: Yoda image courtesy...Check Out Angela Depace's Talk on "Precision and Plasticity in Mentoring" A recent post on the blog “Tenure...

.... Kutubuddin Molla, investigator at ICAR-Central National Rice Research Institute. When it comes to genome...Kutubuddin Molla is an investigator at the ICAR-Central National Rice Research Institute in India. He specializes...soccer ball, TnpB is a baseball — much smaller, but still powerful. Our lab recently harnessed TnpB from Deinococcus... and guide design rules.   Step 3: Vector Digestion Digest either pK-TnpB1 or pK-TnpB2 with BsaI....plasmid is now ready for Agrobacterium-mediated, biolistic, or PEG-mediated transformation into plant cells... genome editing vectors. If you have specific questions, feel free to contact the Molla Lab (X account...https://doi.org/10.1111/pbi.14416  Maher, M. F., Nasti, R. A., Vollbrecht, M., Starker, C. G., Clark, M...

...publication at Addgene. Walton RT, Christie KA, Whittaker MN, Kleinstiver BP (2020) Unconstrained genome ...upstream of either NGA or NGC PAM sequences (Kleinstiver et al. 2015). From these screens, they identified...off-target activity (Hu et al., 2018).  In 2020, the Kleinstiver lab reported the development of near-PAMless .... Furthermore, non-SpCas9's may have other characteristics that make them more useful than SpCas9. For... in target genes resulting in the formation of “sticky ends” rather than the blunt ends created by Cas9...which makes it easier to target AT-rich genomes (Kleinstiver et al., 2016). Type VI CRISPR systems, which ... plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng...

...organization also known as external collaborations. Interestingly, the ability to work with others outside one...important as more complex approaches to scientific questions become popular. There are only so many methods... in Strategic Partnership Management, a very interesting and exciting part of my work. However, this is...collaborations. During my graduate studies at Karolinska Institutet I started working with other groups. First within...minutes. Get to know your collaborators from other institutions personally on conference calls and make sure...opportunities so just pick the one that you feel enthusiastic about and get started. You never know where ...collaborating with your neighboring lab, you've met interesting people at conferences and you have successfully...

...Resistance can be intrinsic, meaning a basic characteristic that naturally occurs in the bacterium, and...but has become resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma... very skilled at the delicate art of survival. Existing virtually ubiquitously in the environment, there...infecting bacteria can take 24 hours or more, and testing for resistance often takes an additional 18-24 ...available. For routine infections, like strep throat, testing for antibiotic resistance isn’t commonly performed...infections is so rare. When antibiotic resistance testing is requested clinically, this involves growing ... antibiotic resistance?  The problem with this question is, unless an antibiotic targets an essential,...

...hybrid guide to be effective. Which bases can be substituted? A guide RNA has many parts. The ~20 base “spacer...dramatically reduce CRISPR efficiency, and DNA substitutions don’t fare much better: they largely abolished...recognizes the 2’-OH of RNA in this region, and DNA substitutions dramatically reduced cleavage (Kim et al., 2020...some parts of the scaffold sequence could be substituted without significantly reducing genome editing...scaffold sequences have different tolerances for substitution of DNA bases. Created with BioRender.com. ...matches, the complex is stable enough that the DNA substitution doesn’t disrupt it. Combined with the reduced...access these kinds of tools. Location of DNA substitution Similar on-target efficacy Reduced off-target...

...025-09021-y  Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020). Unconstrained...whirlwind of developing cell and mouse model systems, testing base editing variants, and assessing the safety... mutation. To tackle this problem, Benjamin Kleinstiver’s lab engineered new variants of S. pyogenes Cas9...came out on top. Narrowing PAM preferences The Kleinstiver lab also worked on the opposite project: engineering...tested mutations produced efficient editors, the Kleinstiver set performed best when paired with the V106W... in different patients. While these gRNAs would still need to be optimized and assessed for off-target... J., Butcher, R. M., Pacesa, M., Smith, B. K., Christie, K. A., Ha, L. L., Meis, R. J., Clark, A. B., ...

..., an individual experiences progressive muscle wasting leading to death at around 30 years of age. Despite...the amount of research conducted on DMD, there is still no good treatment. The dystrophin gene is very large...approach. Since shorter forms of dystrophin can still be functional, exon skipping is a good option for...non-homologous end joining (NHEJ) pathway. It’s estimated that very little dystrophin correction (about ...could make a huge difference in DMD, and it’s estimated that exon skipping therapies would be applicable...experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar to the ...CRISPR therapy closer to the clinic, much work is still needed. First, CRISPR delivery must be optimized...

...to non-jargon in the context of disease testing, it can still be considered jargon in most other uses....situation rather quickly!  The question   The first, and most important question, you should ask for any science...ll see throughout the posts and introduce *the* question underlying all communications efforts.  Scicomm...scientific or science communication. A Tweet is still considered scientific communication if the language...An audience can be defined by any number of characteristics. Common ones include age range, familiarity...patients or caregivers affected by the disease in question who are involved in online patient/caregiver communities...

... preparations undergo rigorous quality control testing at Addgene meaning they come ready made to accelerate...researchers lamenting the viral production process while still ordering viral vectors. Virus production can be ...interested in faster by producing, titering, and testing viral preps for them. At the same time, we’re developing...base for the processes of viral production and testing so that researchers can use our educational resources...researcher Connie Cepko has been helping us with AAV testing and recently had this to say about the service:...suggest additional plasmids with the Viral Service Suggestion Form. Lentivirus As some of the most popular ...well as other expression data. If you have any questions about the Viral Service or any of the materials...