...social media, you could be sharing your research or finding opportunities to bring science to the community...the same page with the potential PI in terms of mentoring style and expectations. Make sure you will enjoy...mantra and never forgot it. Usually when I see inspiring artists who are thinking about getting into the... the process down into steps and set aside time during the week to tackle each one.  Get the toughest ...

...Westerns are divided into two categories, native and denaturing. In a native western, the protein’s secondary...is separated through a matrix by charge. In a denaturing western, the protein is denatured to its primary... purpose of this blog, we will be focusing on denaturing westerns.  Separating a protein mix by size Sodium...The wet transfer process is very effective in transferring a broad range of protein sizes but takes hours...blocking the membrane  from binding to the antibodies during the stain. The ideal blocking buffer depends on...loading control when assessing protein abundance or comparing samples across lanes.  For a typical loading control...

...relatively simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent...Megamer service tested by Quadros et al. When delivering the ssDNA template and CRISPR machinery, Quadros...facilities for 13 projects, with most projects requiring only 40-60 zygotes to generate a correctly edited...conditional alleles by CRISPR/Cas-mediated genome engineering." Cell 154 (2013):1370-9. DOI: 10.1016/j.cell... a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 19 (2018... Mary Gearing...

...year, graduate students and post-docs alike are wondering where the time has gone. Are we any closer to ...to graduating, publishing that key paper, or figuring out a career path? Many trainees are developing Individual...accomplishments, from learning new techniques to finally figuring out a tough experiment, sets a positive and encouraging...Good Manager Anyway Developing Transferable Skills During Science Training ... Mary Gearing...

.... pombe. The protocol is surprisingly simple, requiring only a PCR product containing the modifying sequence...affinity purification of protein complexes. John Pringle and Jürg Bähler have deposited a large collection...genome modification in yeast developed by Dr. Pringle’s former lab at UNC Chapel Hill. Bähler et al.(2...McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR. Yeast. 1998 Jul;14(10):943-51. Longtine MS,... DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR. Yeast. 1998 Jul;14(10):953-61. Note: Marcy...

...openness and sharing in science.  We’re excited to support the neuroscience community by sharing not only ...capsid proteins that determine the AAV serotype. During transduction, the viral capsid proteins bind to...enhancing anterograde transport efficiency, and engineering viral capsids that target specific cell types...science, we aim to accelerate the rate by which sharing speeds science.   Check out the precise neuronal...

...believing, as we do, that sharing speeds science.  This year, Chonnettia will be sharing more of her perspective...what we accomplished in 2023 and thrilled to be sharing our plans for 2024.  In 2023, Addgene distributed... of innovation and impact made possible by the sharing of tools for research: discovered and developed...you a healthy, productive, and thriving 2024, mirroring our own aspirations for Addgene. On behalf of ...

...new technique, developed in Thoru Pederson’s lab, brings us one step closer to mapping the 4D nucleome, ...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...may be imaged under lower illumination settings, lowering photobleaching and phototoxicity concerns and ... of chromosomal translocations or chromosomal shattering (chromothripsis). As this study joins the pantheon... Mary Gearing...

...DCL) process the dsRNA into specific types of interfering RNAs.   RNAi vs. CRISPR: Complementary techniques...fundamental steps: creation of the RNAi trigger delivering the RNAi trigger characterizing the RNAi phenotype...from a long hpRNA trigger, only a single ~21-nt interfering RNA is produced using shRNA/amiRNA. Although ...evaluated for their gene silencing efficacy, thereby requiring a substantial time investment. Once the target...widely implemented in plants, such as in the model flowering plant Arabidopsis thaliana (Li et al., 2013; Zhang...Gong B-Q, Guo Y, Xu L, Zhang X-N, Li J-F (2018) Engineering Artificial MicroRNAs for Multiplex Gene Silencing...

... project. Dissemination of the knowledge gained during this period occurs infrequently and rarely makes... wondered how we could make the insights gained during this time more visible and thereby have a positive...reviewers of your expertise and insight. We recommend sharing ReFigures on your Linkedin and ResearchGate profile...Please support ReFigure by creating ReFigures and answering this 2 minute survey! You can also find ReFigure...science at We Share Science Learn more about Data Sharing Resources at Addgene.org Deposit Your Plasmids...

... the importance of leaving room for serendipity during your PhD studies and offer two simple steps to ...you to discussions beyond your immediate field, fostering a diverse range of perspectives and sparking innovative...capture fleeting thoughts and connections that emerge during moments of inspiration. Additionally, journaling...enrich the depth and creativity of your work, while offering mental health benefits by encouraging activities...How to Lead a Great Meeting Form Your Own Peer Mentoring Group...

...thought leader in scientific sharing, leading by example to increase sharing in the community Addgene fosters...organization what do you think of? Maybe the term brings to mind a social service organization like the ...believes there is strength in diversity and in empowering team members to take initiative and give their...our mission to accelerate research with easier sharing and collaboration. Joanne and all the Addgenies...

...enables researchers to silence or incite neuronal firing and study subsequent effects on behavior. The system...activation of light sensing proteins. Furthermore, during the first 2 weeks of life, larvae already exhibit...the cell membrane when exposed to light. Neuronal firing depends on these charged particles and whether ...said to be hyperpolarized. The chloride pump NpHR brings in negative chloride ions which hyperpolarize the...functions as a neuron “off switch.” Because of the differing peak absorption wavelengths and light intensity...

...used to spend my days (and often my nights :D) engineering E. coli to produce biofuels in Pamela Silver’...enterprise or that there weren’t some pretty hefty bumps during my graduate training. I particularly think that...and interesting than that will simply get people ordering plasmids. The need to keep content coming also...that I thought about careers outside of academia during my graduate training. PhDs have many roles to play...different fields. If you start thinking about preparing yourself for a career while still in graduate ...

...my recommendations on some ways to get started sharing your science with the community. Some of them you... up your own twitter feed or talking about and sharing some science on the one you already have. Read ...University. At Addgene we're committed to scientific sharing – both your plasmids and your research stories....  sharing your science... scientific sharing...

...makes sense that scientists, many of whom are venturing into genome editing for the first time, have lots... the growing PubMed article list, the Genome Engineering Using CRISPR/Cas Systems has expanded out of ...wildtype or double nickase for my CRISPR genome engineering experiments? A1: When assessing which nickase...nickase type to use for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized...amazing resources for the scientific community and sharing these resources with Addgene. Thank you, Le!   ...

...on this topic), for this second entry in the “Mentoring for Scientists Series”, let’s focus on how to ...School researcher Connie Cepko to learn about her mentoring style. How can I find out if an advisor is a good...have good observations and be less inhibited in sharing concerns. Ask alumni of the lab. Most labs have...Depace's Talk on "Precision and Plasticity in Mentoring" Don’t ignore warning signs If lab members, neighbors...a Good Mentor?" and 6 More FAQs About Science Mentoring ...

...gene expression have similar drawbacks, often requiring the destruction of plants or tissue for GUS staining...show the effects of the mutation. But ¼ of the offspring from these plants will be green, containing the...removed to prevent further editing, then green offspring can be selected instead of red ones. Other innovative..., Sun, H. et al. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic...Additional resources on the Addgene blog  CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9 Tips for Arabidopsis...

...dopamine affinity, with 113050 offering high apparent affinity, and 113049 offering medium apparent affinity...target photostimulation of axons and dendrites on neighboring neurons. We have also packaged hChR2(H134R) plasmids...norepinephrine Neuroscience research relies on accurate monitoring of neurotransmitters and we are excited to offer...of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...

...lamenting the viral production process while still ordering viral vectors. Virus production can be a frustrating...experiments they’re interested in faster by producing, titering, and testing viral preps for them. At the same... service: "I'm excited that Addgene is now offering virion preparations to the scientific community...involving CRISPR and so we also have lentivirus for delivering control shRNAs, GFP, and rtTA from the Tet-on...continue to develop it. More communication and sharing will only help research advance. Remember: productivity...

...peer-review is a touchstone of the scientific method, ensuring that published work meets the accepted standards...or omission from the literature. I think that delivering constructive critique can help your colleagues...following morning - as usually deadlines are pressing. During this second read, I pay more attention to details...passionate about open science, BIG data analysis and sharing whatever she knows with whoever cares to listen...the Addgene blog Find blog posts on scientific sharing Get tips on writing a review article Resources...

...our eyes open for any new technologies that will bring about the next revolution in biological research...solution. 5. Cpf1: A New Tool for CRISPR Genome Engineering While the vast majority of CRISPR plasmids in...DNA, Addgene Science Communications Intern Mary Gearing explains how Cas9 homolog, Cpf1, may make it easier...difficult to use with CRISPR as the cargo. Mary Gearing explains some of the clever ways to successfully...validation process for CRISPR mediated genome engineering. 10. Plasmids 101: Control Plasmids Need help...

...requires an immense amount of self-knowledge and an unwavering devotion to seeing your ideas become something...CEO of miLab, a Boston-based startup focused on empowering early-stage scientists. You can think of miLab...generation companies.  We launched in the early spring of 2015 and since that time have received about..., including one led by a talented biomedical engineering team from MIT.   I would like to share my own... are everything and you want to leave people remembering your potential as an entrepreneur, not your credentials...

... 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien, 1999), monitoring kinase ... visualizing structural changes in the ribosome during protein translation (Ermolenko et al., 2007). All...acceptor molecules are targeted nearby (such as a neighboring house on the same street as the donor), then ...amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural amino acid...Cy3NTA and Cy5NTA bind His tags similarly but with differing R0 values, thereby enabling calibrated FRET distance...

...advancements include work exploring far-red-emitting dyes in bacterial imaging, offering solutions to overcome...Photostability, the ability to retain fluorescent intensity during exposure to light, is another factor to consider...wavelengths, as this is limited by the filter cubes and filtering settings of the equipment. Systematic evaluation...facilitate clear differentiation between multiple dyes during analysis. Image resolution is impacted by several...Bhattacharya, K., Pyrgiotakis, G., Zhang, Z., Purington, E., Gramlich, W., Grondin, Y., Rogers, R., Bousfield...

... when scientists bring biases to research and don't collaborate with others to bring diverse perspectives... rules all, the books center around a cast of endearing characters who decide to take matters into their...dueling magicians in the 1800s in England who help bring back actual magic (which had been lost for an age...a book club would force me to read some books exploring new genres, new authors, and new themes. A book...

...roles in fluorescent protein development by openly sharing their resources with the research community. To...protein abundance, possibly due to misfolding. But measuring variants one at a time is time consuming and unscalable...use the sensor to answer the question of whether firing of serotonergic neurons (measured using a red calcium...sensor in sleep-related studies and in assays for measuring human serotonin transporter activity. Unger also...dynamics with the TIMER growth rate bioreporter. During protein maturation, the bioreporter spontaneously...

...a producer cell line like the widely used 293T. During vector production, assembled AAV vector particles...released from the producer cells into the supernatant during the production phase. However, this has not been...at the AAV vectors that are released from cells during production (Maguire et al., 2012). They saw that...: PMC4104587. György, Bence, et al. "Rescue of hearing by gene delivery to inner-ear hair cells using ...of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...

...recombination-mediated genetic engineering (find more information on bacterial engineering using CRISPR on our blog..., their interactions and information processing during this condition is crucial. Plasmids expressing ...interactions. Combining synthetic biology with engineering: glowing bacteria and a fish robot to detect ...interesting combinations: biomedicine and astrobiology. During the last 11 years he has studied various aspects...

... HeLa cells?), genome engineering and stem cells, and the difficulty of sharing banked iPSCs across international...years, I've been looking forward to seeing how the sharing of his OSKM factors have affected the stem cell...paper as "life changing". Dr. Yamanaka himself, during his keynote address, appeared almost incredulous...into a pig. In stark contrast to recent genome engineering technologies, stem cell techniques are neither...

...for various scientific or engineering applications.   References Refactoring and optimization of light-switchable...photobiology: why use light? In synthetic biology and bioengineering, scientists seek to directly probe and build... publication at Addgene. Synthetic biology: engineering Escherichia coli to see light. Levskaya A, Chevalier... Mary Gearing...

...to the interview here! Doench majored in history during his undergraduate work at Hamilton College. When...that was open ended... it was actually something bordering on research, rather than 'here’s the protocol,... He first heard about RNAi at his MIT interview during a lecture given by Phil Sharp. After a few rotations... easier for people everywhere…. the nonprofit, sharing ethic that Addgene has behind it is something that... sharing...

...job. How can you incorporate the skills developed during graduate school into your resume? How can you stand...Here are a few things to consider when you're preparing your resume for a non-academic position: What ... current job as a Communications Coordinator. Wondering how you can develop more transferable skills? ...? See Developing Transferable Skills During Science Training and So You Think You have Skills.   Get Science...services, but some of the general knowledge about preparing a good resume, writing a concise cover letter,...

...off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity is... the Cas9 nuclease. This can be determined by measuring the indel frequency at a target site. A specificity...not be accessible by wild-type Cas9, due to its stringent PAM specificity. This can be especially important...PMCID: PMC6368452. Strecker, Jonathan, et al. "Engineering of CRISPR-Cas12b for human genome editing." Nature...Repurposing CRISPR-Cas12b for mammalian genome engineering." Cell discovery 4.1 (2018): 63. PubMed PMID:...

...” has me considering the microbiome more often than not). If you’re someone who’s considering getting ...Even if you're currently a synthetic biologist engineering E. coli metabolism, maybe you’ll be able to apply...because they provide a highly versatile means of delivering genes to many different cell types with minimal...Optogenetics, Neuroscience, and the Future of Neuroengineering Primer on Optogenetics Resources at Addgene.org...

...interest in sharing, there will always be an interest in protecting intellectual property. Figuring out that...of Katz and Paratore onto the Board of Directors brings complementary experience and insight to the now... to the scientific community through Addgene’s sharing platform. Expanding further beyond the US and increasing...Internal Audit function and is responsible for bringing a systematic and disciplined approach to evaluating...

...Before I started writing for the Addgene blog, sharing Chemistry Cat memes was how I used social media...professionally, and 2) I needed to start using Twitter. Sharing Chemistry Cat memes use to be how I used social...conference, whether you are attending in person or monitoring that conference via social media. The podcast...Twitter is a tool worth using, but you might be wondering how to get started. This blog post outlines 10...unfollow those who do not capture your interest during that week.” You can also find Dr. Knoepfler on ...

...genome replication, repairing mismatched basepairs or small indels that occur during DNA synthesis, and...restriction enzymes are one half of naturally occurring restriction modification systems that prokaryotes...DpnI is often used for site directed mutagenesis. During this process, incorporation of a desired mutation...methylation Finally, you can control methylation by altering your choice of bacteria. For example, if you must...

...components are still there in real life.  Certainly referring to any DNA within the context of a nucleus as ...methylation status at a double-strand break that needs repairing. They may be looking at chromatin at the level...! And if you study chromatin and want to start sharing your plasmids through Addgene, don’t hesitate to...Robert Kingston Thoru Pederson Phil Sharp Julie Ahringer Alexander Stark Bas van Steensel Richard Young...chromatin and the epigenome. If you have been considering some new techniques, the posts below may steer...

...accessibility is removing barriers when we can and lowering them when we can’t. We know barriers exist in ...layers are blurred after centrifugation and the syringe needle is inserted bevel-up at the interface between...the 40% layer. To collect purified AAV, insert a syringe at the interface of the 40% layer containing the... iodixanol layer, with the bevel pointed up. A syringe at the top of the tube allows air to enter the ...the column so the virus will flow out the bottom syringe."Feel free to share your alt text in the comments...

...most important step in ensuring quality, as it will catch any errors made during the cloning, sample handling...One of the best things about sharing plasmids through Addgene is that we provide an added level of confidence...samples coming through our doors, the first step to ensuring plasmid quality is to stay organized. Here at ..., antibiotic resistance, restriction sites used during cloning, and most importantly, the predicted full...

...retinal ganglion. Rather than using naturally occurring promoters to drive transgene expression, the Roska...one type of cell, with the remaining expression occurring in only Müller glia cells, a common contamination...retina.  Find the AAV synthetic promoter plasmids! Comparing synthetic promoters performance in mouse, monkey...targeted cells in monkey and human retinas. By comparing the performance of promoters across these three...be paired for targeted expression. This same pairing of synthetic promoters were also used to target...

...completely different than what was originally intended. Sharing unverified plasmids can lead to far reaching consequences...standardization provides security for requestors, ensuring that the plasmid they ordered contains the correct...QC topics that may require additional attention during analysis. These include topics such as: locations... features. Most of these regions are identified during the QC process, following sequencing and assembly...30% of deposited plasmids! We are committed to ensuring that we maintain a high standard of sequence accuracy...

...made of protein-coding genes, and you might be wondering what the rest of our genome could possibly be ... modulate ncRNA expression without permanently altering the DNA, CRISPR interference (CRISPRi) and CRISPR...high specificity, particularly in applications requiring smaller delivery systems (Zibitt et al. 2021).... a number of different options. By carefully considering your ncRNA of interest, you should be able to...RNA-targeting CRISPR enzymes for transcriptome engineering References Jinek M, Chylinski K, Fonfara I, Hauer...

...their promises, Wet-blanket negativist, who can bring down the morale of a group with their pessimism,...communicate their ideas confidently while also considering the needs of other people. Some students and ...Regardless of your supervisor’s management style, bring challenges to his or her attention as soon as you...signed, or manuscripts that need to be reviewed, bring them to the meeting. This will make it easy for ...problem by stating the facts and explain how it is interfering with your work. Avoid talking about your emotions...

...something done not by people wearing a lab coat, but instead by people wearing business suits discussing ...transferable skills while working in the lab and by offering advice on areas where you might need some help...you are negotiating. Then take a moment before, during, or after a negotiation to think about the situation...experience in the area I want to explore while also ensuring that my work gets completed? I want my company...

...special considerations. Conventional flow cytometry During conventional flow cytometry, light emitted by a...distinguish these two fluorophores from one another during spectral unmixing.     Figure 2: The ...a three color panel or a 40 color panel! When preparing a spectral flow experiment and panel, there are...telling exactly how many Cy7 molecules are added during that particular batch. This can present particular...upon antibody binding, leading to discrepancies during unmixing. Additionally, it is critical to include...

...perform functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided... endonuclease NgAgo! We'll discuss how NgAgo is faring with users in a minute, but, to start, let's review...cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering methods have their own pros and cons (2). The...protein to load an ssDNA guide. It is possible that, during translation, only a small fraction of nascent NgAgo...Development and applications of CRISPR-Cas9 for genome engineering." Cell 157.6 (2014): 1262-1278. PubMed PMID: ...

...Introduction There are three primary methods for repairing DNA after a double-stranded break. HDR copies ...mutations seen with TALENs and CRISPR. MMEJ is active during M and early S phases, when HDR is inactive, and...Non-homologous End Joining Read About Other Genome Engineering Tools like Sleeping Beauty Mutase Find the Ideal... genome engineering... Mary Gearing...

...heterozygote into a homozygote and ensuring that all future offspring inherit the endonuclease gene. As...As we pointed out last year2, delivering a DNA cassette encoding the cas9 gene and single guide RNAs (...shared by everyone, he is deeply concerned with ensuring that gene drives and other collective technologies...selfish genes as tools for the control and genetic engineering of natural populations. Proc. Biol. Sci. 270,...