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...the Wyss Institute for Biologically Inspired Engineering. CRISPR/Cas9 technology has revolutionized the...the fields of molecular biology and bioengineering, as it has facilitated the development of a simple ...relative nuclease efficiencies as well as to more stringent targeting rules that result in a decrease in available... as a means of modulating gene expression (6). Altering the length of gRNAs targeting different sites ...Santa Cruz, where her research is focused on engineering nanoparticles as drug delivery systems. She is...develop several popular CRISPR-based tools for altering DNA sequence or regulating gene expression.   ..., Raj, et al. "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach....

...make genomic alterations, bringing about a revolution in genome engineering, with new techniques rapidly...and can be used for most of the current genome engineering applications of CRISPR including: generating ...delivery. You’ve decided to use RNPs, now what? Preparing the Cas9-gRNA ribonucleoprotein The first step... the Jacob Corn and Alex Schier laboratories. Delivering Cas9-gRNA ribonucleoproteins  An advantage of...applications. This technique utilizes Cas9 proteins harboring receptor ligands (C in the figure above), which...stable in human plasma than the standard spCas9 (Harrington et al. 2017). Lastly, as with any scientific ... Addgene.  Andrew's interests include genome engineering and microbiology.     References 1. Chen, Sean...

...proteins in solution. The pH is then lowered using a renaturing solution, which causes the proteins and genomic...purification and DNA gel extraction with glass syringe filters Starting material: bacterial cultures or...binding DNA to silica columns, it’s bound to glass syringe filters. Normally DNA does not bind silica or glass... kit takes 1-1.5 hours to complete, while this syringe-based method can be completed in 20 to 30 minutes...culture you’re working with. Overall, the glass syringe filter method is not only cheaper, but also faster...gel slices Product: DNA fragments You might be wondering, “How is milk going to help me extract DNA from... 48 kb with high yields and without degradation during the extraction procedure. Additionally, purified...

...accelerating scientific discovery. Over 150,000 plasmids empowering researchers worldwide. Countless breakthroughs... to make it easier to find what you need and to bring it in line with current Web Content Accessibility...contributions to key research papers are already inspiring higher standards in antibody use (Ayoubi et al...packaged on demand in five distinct serotypes, offering you unparalleled flexibility. But this is just...characterization data for over 150,000 plasmids, empowering bioinformaticians, data scientists, and developers...collaboration formed, and every breakthrough enabled brings us closer to making an impact on humanity's greatest...this incredible journey. Together, we're not just sharing research materials — we're accelerating the future...

... Members periodically break, go exploring outside the team, and bring information back. No team has all...contribution short and sweet. One key to this is ensuring that the group always conducts exceptionally well-managed...manager can be efficient without delegating and empowering the team to solve problems together (tactics ...important paths of information flow is the one that brings feedback back to the manager. Team members rarely...often A one-time event doesn't work to get people sharing.   Frequently ask direct questions for specific...at formal communications events or at informal gatherings, like at lunch. Many experts recommend using ...constructive feedback and suggestions. Consider offering rewards (gift card, afternoon off, etc.) Act on...

...transferable skills while working in the lab and by offering advice on areas where you might need some help...important in a non-research context, especially when considering small to mid-sized companies and organizations...time I’ve had a role in organizing collaborations. During my graduate studies at Karolinska Institutet I ...what was expected, and keeping track of speakers. During your graduate or postdoctoral work there are loads...you develop will lead! Read our blog post about fostering communication in remote collaborations! Stay in...Great progress! You are collaborating with your neighboring lab, you've met interesting people at conferences...twitter and maintain a connection by commenting and sharing. LinkedIn or ResearchGate allow you to send personal...

... your foot in the door When I was first considering preparing for and transitioning to a career in healthcare... Healthcare consulting is a fast-paced field, requiring people who are willing to quickly learn, and apply...governments on the most effective and efficient ways of delivering healthcare. Clients typically engage consultants...fields. I got my PhD a couple of years ago in Bioengineering, and one of the things that pleasantly surprised... suite of benefits a given therapeutic approach brings to patients, doctors, and insurance providers, ...aspect of research in biology or medicine you will bring an essential set of tools to a consulting team. ...an increasing recognition of the value graduates bring to these roles. Healthcare consulting is a demanding...

...Read here to get help if you are having trouble preparing your most excellent resume. 2) Follow job boards...reviewed regardless of personal connections to the hiring group.  Read here for more on timely submission... job description is usually a wish list and the hiring manager knows that no applicant is likely to meet...Your resume must be in the official “system” for hiring managers to take next steps and follow up especially... as you will not likely know the exact person monitoring submissions (usually a Human Resources representative... your resume pdf document. 6) Use your network! During the entire process, use whatever means you can ...can to get your resume into the hands of the hiring manager on paper or as an email send directly from a...

...C., Addgene’s intern coordinator shortly after hearing her talk about Addgene’s internship opportunity...summer interns presenting their capstone projects during the Summer Intern Poster Presentation.  As ...the Syntaxin3 gene in a pLenti vector backbone. During those five weeks, I worked with my mentors, Katherine...always came with surprises. Originally we thought during the first three weeks that we had made a mistake...not having the right concentration of a reagent during our HiFi assembly but decided to go ahead anyway...restart the procedure we would not have enough time. During the following two weeks, we were able to obtain...on my capstone project report and presentation, during the final week at Addgene. This report shared my...

... assays are not with denaturing agents like DTT or β-mercaptoethanol.  Denaturing proteins To denature...blot, your loading buffer will not contain any denaturing agents. However, you’ll still have to lyse your... post, we are assuming that you are running a denaturing blot.  There are a number of lysing methods and... aliquot your sample(s) before adding in your denaturing and/or loading buffers, as most Bradford assays... proteins in different patterns, due to their differing chemistries. You therefore cannot compare Bis ...and reprobed multiple times. It doesn’t degrade during long-term storage and has both high binding capacity...well for proteins of most sizes and are worth considering if your proteins of interest vary wildly in size...

...research purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and ... host’s genome isn’t uncommon. Many naturally occurring viruses occasionally integrate their genomes into...the host genome via the viral proteins packaged during production (read our blog post on the retroviral...time, IDLVs are ideal. As the episomes are lost during cell division, the expression of the delivered ...production protocol.  One thing to keep in mind is that titering these IDLVs may require a slightly different protocol...regular lentiviral vectors. This is because most titering methods rely on isolating genomic DNA from transduced...DNA along with a reference cellular gene. When titering IDLVs, there is no integrated piece to measure...

...be taken seriously so we made it official by registering BosLab inc. as a nonprofit organization in the...are skilled with audiovisual media, software engineering, business consulting, mathematics, and more. ... to perform proof-of-concept experiments for discovering novel therapeutics. Dave is a chemical biologist...and in his free time, he is pursuing a plant engineering project at BosLab. Once he optimizes plant cell...knowledge with the community by teaching a DIY plant engineering workshop. Community projects at BosLab Not all...and Mrina Nikrad have started the Open Yeast Engineering Project (OYEP). Through this project, community...do similar things. The focus is to make yeast engineering accessible to everyone but these projects could...

...problem solving. I believe that is something lacking during degree courses; they never really prepare you for...failure and problem solving scientists experience during research. Working abroad in rural Africa Once this...another area which I was really passionate about during my degree. I loved parasitology. However, when ... held me back; I do not have a second language. During the recession there were many qualified scientists...Genetics to give me an edge over other graduates during the recession. As each advertised job received ...the training and experience relating to good manufacturing practice (GMP) and good lab practice (GLP) (...recognised the logo, but didn’t know much else. During the interviews, I asked if I could contribute more...

...head when you hear the phrase “action potential?” Firing neurons are likely the first thing that comes to...reprogrammed regenerating tissues without using genome engineering. Planarian worms cut into three segments “remember...communication is briefly inhibited in the entire worm during regeneration, the worm creates two heads. Subsequent...resources from the bioelectricity field In addition to sharing tools to study bioelectricity, bioelectric researchers...defects, regeneration, medicine, and synthetic bioengineering. References Chernet, Brook T., et al. "Use...growth and form." Annual review of biomedical engineering 19 (2017): 353-387. PubMed PMID: 28633567. Mathews...bioelectricity for regenerative and synthetic bioengineering." Current opinion in biotechnology 52 (2018...

...open science nonprofit that would facilitate the sharing of plasmids between scientists. The service would...resource. I really appreciate the work you do to make sharing reagents and protocols so easy!” — Nina Jain, Harvard...seven before we had 1,000 depositing scientists. Ensuring that people knew how to access Addgene, and that...2012, our repository was rapidly able to support sharing of CRISPR constructs and information across the...lentiviral and AAV vectors, as well as developing and sharing supportive educational resources. By 2017, we had...NGS run.   To twenty years and beyond! Which brings us to 2024, Addgene’s 20th anniversary and the ...been made because scientists believe in openly sharing their plasmids. It’s been a privilege to find and...

...numbers of women participating in science and engineering. “Fix the Institutions” promotes gender equality...billions—but when drugs fail, they cause human suffering and death. Doing research right has the potential...between male and female cells. The result of not considering the sex of the stem cells was that their male...sex, and it’s important to analyze these factors during an experiment. In the case of stem cells, these...tissues—when that information is reliable—is important to ensuring excellence in basic research.  In addition to ... the sex of the most commonly used cell lines appearing in American Journal of Physiology-Cell Physiology...clue or potential place to start research is by considering sex disparities in disease severity. Such disparities...

...wash buffer such as PBS or TBST. Stringent stripping The stringent stripping solution uses heat and detergent...your sample? Or what if something weird happened during your western and your results look… funky? Do you...along with the antibodies, especially with more stringent stripping methods. If you are planning to detect...that section of the blot unusable for reprobing. Preparing your western blot Run your gel and membrane transfer...primary and secondary antibodies from your membrane, clearing the way for the second blot. There are multiple...with a mild stripping solution and move onto a stringent solution if the mild is not effective. You may...perhaps using longer incubation times, or try the stringent solution if you started with the mild one. Reprobe...

... steps, until its connection with DNA is broken during cross-link reversal (not shown in diagram). Cross-links...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original CAPTURE...) one or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and...detecting chromatin interactions opens the door for answering more questions about chromatin conformations. ...‘social networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions A third...in chromatin conformation, like those that occur during development and cellular differentiation. Using...the Xu Lab tracked enhancer-promoter interactions during erythroid differentiation. CAPTURE 2.0 captures...

...of choice is AAV, with nearly every experiment requiring AAV. Before joining my lab, I had never worked... a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too hot because.... Rather than investing all of this effort in preparing reagents, I choose to only harvest the cell pellet...layered and then the viral suspension generated during AAV production is overlaid. Following ultracentrifugation...first purification. This helped me get a feel for layering the iodixanol solutions and let me double-check...make one or two small aliquots to use for qPCR titering. This helps avoid multiple freeze/thaws of your...of virus. AAV titration by qPCR Overview: AAV titering by qPCR quantifies the number of genome-containing...

...packaged inside the vectors during production. These impurities cannot be removed during the purification step...recombinase-independent recombination during plasmid growth in bacteria or during viral production. This recombination... control assays for AAVs. These steps include measuring viral titer and assessing purity. But Addgene ...wrong plasmid? What if bacteria snuck into the prep during the production process? These questions are especially...materials. Addgene is going the extra mile and pioneering the systematic use of this new QC assay to guarantee...Evidence for Encapsidation of Prokaryotic Sequences during Recombinant Adeno-Associated Virus Production and...