...very small homology regions (5-25 bp) for repair, making it easier to construct targeting vectors. Addgene...copies the sequence from a repair template with flanking sequence homology for error-free DSB repair. NHEJ...MMEJ uses regions with 5-25 bp of microhomology flanking a DSB to repair DNA. The DNA ends are chewed back...figure above, short (5-25 bp) regions of homology flanking a double-stranded break yield precise deletion... is likely to introduce additional nucleotides flanking the insertion. If the DNA ends anneal incorrectly...5’ and 3’ microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks...sets of microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus (see figure...

... unbiased potential to find things you are not looking for, but there are also benefits gained by lowering...differentially expressed genes can be the next stage in making sense of such data. These targeted screens can ...ligand (such as when a component of a microbe or a cytokine is added to a cell). With a custom CRISPR screen...example, one could study the cellular response to a cytokine known to cause the up-regulation of an important...cell). In this screen, known components of the cytokine signaling pathway could be targeted. Cells failing...the transferred cells is followed. A benefit of working with surface proteins is also that such proteins...eleven published libraries are embedded. These were kindly donated by six different research constellations...

..., D.C., we must be making a dent in the global coffee supply… My back-of-the-napkin calculations put the...attendees. And for those of you presenting, working, and networking at SfN, remember there are only 2 days ...community pulls off such an inspiring, thought-provoking, collaborative event – all while keeping the party...CRISPR-Cas tools into neuroscience experiments. Speaking of CRISPRs, we were excited to have Feng Zhang...we've spoken with are excited and passionate about making such tools open-access. Though it is time for me...

...knowledge, shaping their trainees’ very way of thinking too. Albert Einstein might have put it best when... (Brian, 1996). But is it possible to bring the kinds of transformative educational experiences afforded...happened in the lab, and he wanted to provide the same kinds of opportunities for his students. But he also found...research capabilities and laboratory and critical thinking skills (Knutson et al., 2010a,b). Classroom-based...student apprehensions about “failure” (protocols not working, hypotheses being rejected, etc.), while reminding...excellent training in improvisation and the latter making him a more effective motivator (“be like Tony Robbins...at other institutions. Provost believes these networking experiences increase student enthusiasm, and ...

...by (as the name suggests) cross-linking cellular proteins. Cross-linking is a chemical reaction that covalently...solutions.  Along with preserving your sample, cross-linking can block the epitope of some targets, preventing...antibody from binding (Fig.1). The level of cross-linking that occurs depends on the incubation time and ...may be able to recover good antibody binding by tweaking those parameters, but in some cases your antibody... good for epitopes that are sensitive to cross-linking, but it doesn’t preserve sample structure quite...decide which ones to use? Here are some tips for making those decisions: Always always always look at ...recovering epitope availability following cross-linking fixation; they require an additional step generally...

...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...facility). The third tube is used to fulfill orders. Breaking the (bar)code At each step in the receiving, transforming... traced. If you’re just starting a lab, or are looking for a way to organize samples within your lab, ...in house if necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes... information in a small space and is ideal for tracking large collections. Many companies, such as Thermo...Although we’ve developed our own computer system for tracking samples, it can be done easily with a barcode ...multiple people or even multiple labs. Careful tracking prevents us Addgenies from wasting hours of valuable...

... paper into an electronic environment and hence making it searchable and shareable, but some ELNs provide...community were to: (1) prevent vendor lock in by making it easy to get data out of the ELN; and (2) facilitate...repositories. Below I describe how each of these kinds of connectivity facilitates reproducibility. Connectivity...references the record of research is incomplete, making it difficult or impossible to reproduce. As the...the graphic depicts, RSpace supports linking to all of the most commonly used online file storage tools...Slack conversations and import them into RSpace. The kind of informal conversations about research that can... are crucial to understanding research and the thinking and workflows that went into it. The ability to...

...growing quickly. Taking what I loved from each of these platforms (such as social networking, visual appeal... This post was contributed by Ryan Watkins, Professor at George Washington University and developer of...average video abstract view counts.” For researchers looking to garner more attention for their work, a video...discuss in-progress research, proposed research looking for funding, or are by researchers for whom English...English is a second or third language (making publishing their research all the more challenging even when...and let the world know about your science! Ryan Watkins is a professor at George Washington University ...the Addgene Blog 6 Tools & Tips: Online Social Networking for Scientists Read about the Evolution of Scientific...

...Harvard, Johns Hopkins, (most of) MIT and the University of Colorado are doing, and are checking with Yale ... salaried workers receive overtime payment for working more than 40 hours per week will increase from ...scientists within academia, most particularly postdocs working in the U.S., whose current salaries are below the...scientists are paid above this threshold or for tracking the hours that these scientists work and paying...Department of Labor issued a Notice of Proposed Rulemaking, soliciting feedback by Sept 4th.  They proposed...across academia). Increased postdoc wages, overtime tracking? What are institutions doing? Institutions are...what their institutions, or institutions they're thinking about applying to, are doing - for example, your...

... most, get pleasure from taking up such complex challenges, quickly breaking them up into smaller achievable...clients. In this realm, there are a few different kinds of challenges that consultants get called upon to...healthcare consultant, I have worked on most of these kinds of projects. Almost all of my projects, have involved... The fast paced nature of projects often means working long hours both at home and at the office. Consulting...firms are increasingly aware of the toll that long working hours and the unpredictability of schedules can... career in healthcare consulting, I found that talking to alumni, friends, and friends of friends who ... an engaging way to learn about the trends and workings of the healthcare industry. Finally, on-campus...

...Addgenies, development includes everything from tracking plasmids from the moment a deposit kit is sent...we are highly collaborative, we love teamwork. Working collaboratively allows our team to best serve Addgene... example of agility at work is our process for working on a new feature. We start with a story that describes...best approach it in our code.  Sometimes while thinking through these steps, we discover we need additional...otherwise, this isn’t how we work. We all take turns working on different parts of the code. I think the best...we might have an EMTA theme and we will all be working in the EMTA area of the code. By having everyone...Modern Greek to be easily accessible today. Roughly speaking, we translated our software from Python 2.7 to...

...similar to that in samples of interest. If you are working with cells that are amenable to genetic knockout...knockdown approach instead. In this approach, you’re looking for an antibody that detects the target in the ...can therefore be helpful to try all three when looking to label an intracellular target.   Figure...background staining. This can include using: different blocking reagents; titration of antibody concentration ...flow cytometry through this approach. If you’re looking for a flow antibody, the YCharOS (Antibody Characterization...there is a cell treatment (e.g. PMA or a specific cytokine) known to either induce or suppress the expression... which may not be available to every scientist looking to do a flow cytometry experiment. But validation...

...know exactly what you are looking for and how many of that thing you are looking for, you are ready to begin..., it’s helpful to know how many copies you are looking for. Secondly, categorize your edit – is it any...that you won’t need to screen hundreds of clones looking for your edit of interest. For all of the screening...population. This way you can ensure you aren’t looking for a needle in a haystack before going on to generate...do. A popular screening method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et... there is at least ~200 base pairs of sequence flanking the editing site on either side of the amplicon... software very similar to TIDE known as TIDER (Tracking of insertions, deletions, and recombination events...

...to the meeting, and its history of code making and code breaking is wonderfully analogous to genetic circuits...our baffled flight attendant called them, "long, skinny things") and generally exuding a very-tired-but-very-excited...as an undergraduate, I never fooled myself into thinking I was an engineer. The talks we heard Monday night...discussed in the book). These synthetic biologists are taking the "genetic code" to a new level, changing it ...years. I kept busy for the whole poster session, talking to people from every conceivable background at ... Eric J. Perkins...

...Joanne's Reddit AMA  Few of us spend much time thinking about conserving resources in the labs. Disposable...Founder of Seeding Labs, tells the story about working as a student in a rice lab in the Ivory Coast where...very environmentally conscious friends who had me thinking about energy conservation even before it was a...accessed blogs on BitesizeBio is the protocol for making your own Taq polymerase by Outreach Scientist, ...founder, Melina Fan, recently shared our protocol for making competent cells to save time and money. My other... portion of the gel.  What a waste?  I started looking for smaller and smaller gel apparatus until I settled... hilarious.  Birth of the baby gel box Finally, taking conservation to new levels, I was bothered by the...

...different kinds of grants that you can apply for. If you are in a lab, your PI is likely working on a number..., we posted these 25 great tips for scientists working at home, and you will still find this useful. Now...been doing this for almost 6 months, you may be looking for more ways to make sure you are moving forward...Addgene have gone virtual!   2. Practice public speaking For almost any science career, there will be a...good progress on both building relationships and thinking about future career options. 5. Write a grant ...Science If you have a method that you've been working on, let us know in the comments below! Additional...

...I’m working with a talented journalist to shine light on living as a scientist in Beijing, talking to ...to people about taking beautiful photographs of their studies, reporting on amazing women in science, or... or asking someone how and why they left academia. Humans are designed to pay attention and enjoy anecdotal...We build narratives around the self, too — when talking to friends or dates or colleagues we tell tales... a solid point to kick off at. Whatever you’re working on, start wherever it makes sense to start and ... overnight. The only way to improve is to keep working on telling a story again and again and again until...

...genome is 1 in every 256 base-pairs of DNA sequence, making it very useful for genetic engineering approaches...transgenic cell lines. The traditional method of making stable cell lines is to transfect, select, and ...negative impacts on fitness. One consideration when making a transgenic cell line with the piggyBac™ system...However, several challenges still remain, such as making gene edits completely seamless (i.e., so no other...piggyBac™ transposase (PBx), which contains mutations making it competent for excision, but defective for integration...containing a negative selection marker such as thymidine kinase, a researcher may subsequently kill off any cells...target sequence. This allows researchers to avoid making unnecessary base pair changes as would be the case...

...distanced work more creative. An example could be making lab meetings more interactive and creating space...interaction and brainstorming. This could be achieved by asking each presenter to come up with a scientific question...: “Science is increasingly done in large teams, making it more likely that papers will be written by several...taste of that casual kitchen chatter, ultimately sparking new ideas. Social no-science-y meetings Set up...probably lost during these weird times of shift working and social distancing. The take home message is...are endless. Let’s adapt and embrace new ways of working, reconnect as human beings and colleagues, eventually...confidence to launch new and diverse careers by taking part in career panels, and volunteering for scientific...

...Addgene, who will be taking over the distribution to expand the network of scientists working on this even more...throw a therapeutic monkeywrench into the virus’ hijacking of human cells. Even better, more than half the...the Krogan lab's Addgene page to get notified!) Attacking coronavirus, one FedEx package at a time Since...interest, because it means more scientists will be working on this problem more quickly. Figure 2: Getting...headache receiving constant email requests and tracking their fulfillment manually in a shared spreadsheet...interested in facilitating collaborative science, and making it accessible to all people, not just scientists...