...targeted region, as shown below.  To identify optimal nickase designs, IDT scientists Mollie Schubert...and Yan next examined a situation thought to be suboptimal for HDR. At the AAVS1 locus, the two nearest ...13 nt away from the target site rather than the optimal <10 nt distance. Employing two gRNA sites that ...’s best advice: Use a PAM-out configuration Optimize your spacing D10A: nick sites separated by 37...in this post were adapted from an IDT webinar: Optimized methods to use Cas9 nickases in genome editing...

...will influence the stability of your sample over time and determine which experiments you can effectively... solution is fundamental, and it will impact the time you will be able to store your sample without having...preserving the stability of your preparation for a long time. Tris buffer controls the pH, while the EDTA chelates...stability during storage over prolonged periods of time, especially at room temperature or at 4°C. Storing...better than water in these conditions.  The next time you do a plasmid prep, plan in detail before you...

...at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a brief comparison...Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome Editing...have increased thermostability also had a longer lifetime in human blood. To test if this was also true ...without the need to grow the organisms at lower than optimal temperatures. GeoCas9’s thermostability could also...Lucas, et al. "A thermostable Cas9 with increased lifetime in human plasma." bioRxiv (2017): 138867. Additional...

... do my internship in the daytime.  Getting into the office for the first time, being introduced to all...on a time crunch and knew that if we were to restart the procedure we would not have enough time. During...I got from the La Vida Scholars tour. After some time had passed, I reached out to Amy with exciting news...

...Tm and primer dimer estimation, and other primer characteristics. NEB Data-optimized Assembly Design (DAD...post-translational modifications and polymorphisms Codon optimization tool: To access this tool, set up an IDT account...Sequence tool.   DNA sequence manipulation tools  Sometimes DNA sequences need to be “cleaned up” or changed...Sharing your lab protocols using apps to save you time and track your experiments  Resources on Addgene.org...

...attempting to put together such a presentation many times, I could never actually figure out what skills were... mastery. Scientists need to be doers for a long time before they master the skills they need to be successful... learners so we are all likely spending a lot of time in doer mode even late in our careers.  Managers...collaborating effectively with other teams. While they sometimes dig in and do some of the work, they must be more...others take the reins. They work alone much of the time, depending on the organization they lead. Effective...

...factors in mind at all times when making decisions, assignments and polices. Sometimes the boss has to be ...harder to manage when the measure of work is not just time spent at the desk, but it is worth it. This requires...Good relationships with clients and colleagues Sometimes this means you have to fire a good performer if...deadlines (there’s that need for flexibility, sometimes taken to an extreme by scientists) Are highly ...

...you spend a good chuck of your mental energy and time. The research project also serves as a stepping ...productivity and direction of the project? What is your timeline? A typical research project can take several years... your project will be fresh as well.” Don't underestimate the importance of choosing a solid research ...proposal before even touching a pipetteman. Take the time to generate new ideas and think about them critically...George Church (Harvard Medical School) for taking the time to speak with us about adding new scientists to ...

... are venturing into genome editing for the first time, have lots of questions. To keep up with all these...experiments, consider that wildtype Cas9 with optimized chimeric gRNA has high efficiency but has been...Zhang lab, which has comparable efficiency to the optimized chimeric design but with better accuracy (in other...similar to using a single chimeric gRNA. At the same time, the off-target effects are reduced because the ... protocols developed by a variety of labs and optimized for specific plasmids Links to different software...

...grafting different parts together. By combining two optimized elements of an antibody probe, scientists can ...some limitations. GFP has long protein maturation times which limits its use for imaging a protein’s entire...them hard to amplify and image for long periods of time.  Are antibody based probes a solution? To address... localization of single HA-tagged molecules over time.  The Stasevich lab also proved that HA frankenbody... a developing zebrafish embryo over an 80 minute time course.  By stitching together a stable scaffold...

...multiple timepoints during reprogramming. These library-specific identifiers act like a timestamp and help...captures the gene expression of cells at a single time point, making it difficult to investigate how early... 10-week period. Their expression decreased over time but they were not silenced.    Find CellTag libraries...tagged with different CellTag libraries at three time points during the 28 day study: prior to reprogramming...

...advancement. Scientists do and should tell stories all the time. They provide accounts of their research struggles...interview question: Interviewer: “Tell me about a time you had a conflict at work.” Interviewee: “I did...for a paper we were publishing, but when it came time to add author details a different person in the ...great example of storytelling – you get context, time, cause, effect, and a solid conclusion. Advice for...possible — if you find yourself jumping backwards in time to explain something, rethink your structure. Develop...

...respect the time your Mentor is giving by following up on plans. Make a commitment to put time and energy... Specific, Measurable, Attainable, Relevant, and Time-bound. It is a good idea to take larger goals (e.g...and record milestones in reaching goals. The more time and thought you put into setting goals, the more... posse holds me accountable for attending and sometimes it is only the knowledge that they will be watching...

... system, each isotype is expressed at different times of the immune response and initiates different immune...occur, known as genetic drift, meaning that over time, the antibody you expect it to produce is not necessarily...antibodies are plasmid-based. Their sequences can be optimized for specificity and sensitivity, for example by...synbodies, can be produced from plasmids, saving time and money. Nanobodies and synbodies are just as ...Enzyme-linked Immunosorbent Assay). Immunoprecipitation Sometimes, instead of just detecting the presence of protein....2017.01589 Ayyar BV, Arora S, Ravi SS (2017) Optimizing antibody expression: The nuts and bolts. Methods...

... misfolding. But measuring variants one at a time is time consuming and unscalable.  Melissa Chiasson,...several rounds of dose and drug adjustments. Unger ultimately hopes that medical professionals can genotype...it study bacterial population dynamics with the TIMER growth rate bioreporter. During protein maturation...simultaneously monitor cell cycle and growth rate using TIMER and an unstable fluorescent protein reporter under...

...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...approximately 20 fold by directed evolution. They then optimized the length of both linkers to generate a reporter...and detected fluorescence within 2 to 5 hours, a time consistent with previous methods. In zebrafish, ...embryo development. GFP fluorescence increased over time and showed a clear pattern in the developing forebrain...Failure for cells to die or cells dying at the wrong time and/or place can lead to an array of diseases and...

...costs, and time away from home. You can choose your level of involvement and how much time you want to... biggest differences: you don't have to build in time between sessions. “At big conferences, you could... upvoted question.”If the Q&A session ran out of time, the speaker could continue to answer questions ...waiting for a session to start. “There was not much time to network,” Zhang says. “The schedule was pretty...

...saves you from having to create a new fly line every time you want to express YFG in a new tissue type (more... new fly with hedgehog under a new promoter each time a new hypothesis is tested. Controlling multiple...running with fly lines that are already available, sometimes you’ll have to make your own. In my next post,...process of generating your own fly lines. In the meantime, be sure to browse the publication references ...expression systems in Drosophila: A synthesis of time and space, Trends Genet. 20, 384–391 (2004). PubMed...

...adjust our estimates based on the journal that published the articles.  That is, we re-estimate the citations...plasmids deposited at Addgene – typically about four times as many as papers without plasmids deposited with... This is also true for other journals, so our estimates now take into account any differences in citations...deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the same journals...

...in his free time, he is pursuing a plant engineering project at BosLab. Once he optimizes plant cell culture...training prior to getting started. For example, Timothy Stiles joined BosLab last year with a computer ...experience. However, after a few months of dedicated time at BosLab, he became our bacterial transformation... we’ve developed community projects. Recently, Timothy Stiles and Mrina Nikrad have started the Open Yeast...

... we hear about issues with reproducibility, time and time again, the problem comes down to inadequate ... the same result. So, then they end up investing time that they thought would be moving forward instead...lucky and manage to escape, but you’ve lost a lot of time. It doesn’t have to be this way. We can never completely...paper is the one that you need to spend the least time on—just put down a basic outline and refer back ...

...meetings, we cut the time down to an hour or less and are able to provide more timely feedback on each other...idea why you are there. Make the most of everyone’s time by considering the level of meeting you need. Project...meetings short, informative, and pleasant. Email: Sometimes, the right meeting is no meeting at all. When ...assigned items to keep the project moving. Use time wisely Meetings are for talking, not reading: Send...

...promoter driving multiple combinations of WT, codon optimized, untagged, cleavage sites (thrombin and TEV), ... coding genes, tags, truncations, and codon optimizations similar to those found in the bacterial expression...plasmids Finally a set of wild type and codon optimized, full length and truncated versions of SARS-CoV... scientists without performing the full suite of time consuming, costly, and laborious QC for all several...re glad to be able to do so during this critical time. Visit our COVID-19 Plasmids and Resources Page!...

...or if you'll be going through small aliquots at a time, the -20°C is your best option, in aliquots no smaller...experiment you’re planning to run with the antibodies time-sensitive, using precious samples, or being compared...reaction. For instance, sodium azide, a common antimicrobial agent added to buffers, can bind to amine groups... of 0.01% w/v can be used as an alternative antimicrobial.  Conjugates And HRP brings us handily to the...for an experiment is easy, but what about those times you need more than one?   Multiplex assays Multiplex...

...are powerful tools to measure real-time cellular reactions to stimuli and as well as changes in cellular...analyte present in the system at a given point in time. For example, if ATP was the molecule of interest... sensors are fluorescence-based and have been optimized for a wide-range of uses to monitor the fundamental...systems, rather than transiently overexpressing at the time of use. Another great viral perk for delivery is...

...the number of genes that can be manipulated at one time. Weissman’s group has made standard protocols available...available and they are working to further optimize those now. They are also developing sgRNAs that will...there is potential there,” he says. "We have to optimize how we can deliver these tools because obviously...mouse, we can’t rely on genetic engineering to optimize humans for CRISPRi and CRISPRa.” Before considering...information about SAM, including a tool for getting optimized guide sequences for activating any human gene,...

...course, real relationships go further and grow over time, but being interested in the same phosphate of your...connections What can you do to get the most from the time and money you are spending on a conference? One ... onsite and eat together because scientists have time to really connect with others whose company they...yourself. Put your phone away! This is a natural time to meet someone since you are both interested in...promised to send a paper, send it. If you had a nice time, send a note to say so. If you read a paper that...

...signal at the same time point. E. coli’s BirA* was tested at its optimum 18 hour time point, but one minute...Sometimes it feels like DNA and protein get all the attention.There are numerous ways to detect DNA-protein... interactions in events that occur on a short timescale, such as during drug action or cell signaling....

...developmental delays or autism spectrum disorder. It is estimated that CTD is the second cause of male intellectual...psoriasis is a chronic autoinflammatory skin disease estimated to affect 0.27 to 4.6 per 10,000 people globally...of the aorta) are two heart defects that are 50 times more prevalent in women with TS. “The goal of our... bicuspid aortic valve at a rate that is 50-100 times higher than women in the general population,” says... the signalosome of different receptors upon stimulation. “To study cellular transduction events, we will...

... research and clinical applications. Most of the time, when someone speaks about an affinity reagent, ...large enough that their bulk can be a barrier at times. Smaller, slimmer affinity reagents can have deeper...have them, you’ll need to either re-develop or re-optimize your assay for an entirely new affinity reagent...reagents you use, one thing is clear: it’s an exciting time to be a scientist looking for that perfect fit!  ...References Grumezescu, A. M. (2018). Drug targeting and stimuli sensitive drug delivery systems. Elsevier Science...