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... guide is typically 20 nts and the genomic locus must often be proximal to a PAM site for DNA cutting ...an elevated mutation burden. The CRISPR platform must be optimized to reduce off-targets and patient risk...downstream. The con to these approaches is that NHEJ must perform the repair to allow for detection, not Homologous...which does not depend on a DNA mutation, off-targets must be quantified slightly differently. For a comprehensive...to control for off-target events Sometimes there just isn’t a way around off-targets. Your experiment ...have flexibility with gRNA selection, or maybe you just can’t get them below a given threshold even when... For some applications, this is ok, but it still must be controlled for and acknowledged as a potential...

...writing posts. Since then I’ve realized my figures are just as important, if not more important, than my writing...professional-grade design software, like Adobe Illustrator, and I didn’t want to pay for these programs ... Vectr is like a pared down version of Adobe Illustrator. There’s both a web and a desktop version of ...Inkscape Inkscape is the most similar to Adobe Illustrator out of the three options for drawing your own...20,000 icons is designed by professional medical illustrators with input from life scientists. It’s easy to...the workspace. The color and size of icons are adjustable. New icons can be requested, although there’s...

...dysregulated water bath boiling your experiments, just about anything can disrupt your bench work and sometimes...infinite regulatory capabilities of gene pathways just waiting to be discovered. As we started building...unlock the potential contained within each. There must be some way to use LEGOs, which are practically ...toys” gaining life and working as scientists in a custom made, colorful laboratory. When we started, the...this point! It was a great treat for us to put our rusty imaginations to use in this world where we are [email protected]. Who knows, your story might just get “legofied.” Many thanks to our guest blogger...

... work in the biotech, education, or nonprofit industries. First up is Hans Packer - science writer  When...an application support/sales representative in industry or a postdoc in academia, but knew that, without...gamble, and it’s paid off. Work is never boring. “I just keep learning on the job. It keeps it interesting...official title may be science writer, writing is just the one of the skills Hans uses for his job. “I ...(get tips for your next science talk). I do illustrations for summarizing mechanisms and applications ...science writers who can get everything that they need just from reading up on a topic, but I haven’t met any...

...often be specific to denatured versions, or even to just that particular assay.      Fig. 1: A native...proteins instead of denatured ones - well, don’t relax just yet. Proteins often change shapes and epitope accessibility...binding to other proteins, changing locations, or even just as part of performing a specific function. A transmembrane...necessarily need to know where the epitopes are, just if the available epitopes could be affected by, ...the assay can help you avoid a great deal of frustration when designing and troubleshooting immunoimaging...protein is doing or changing in different conditions. Just remember to keep good records on which antibodies...

...initial read, adjust my language in the first (and clunky) version of my general comments and adjust them if...Magdalena Julkowska, a postdoctoral researcher at KAUST, Saudi Arabia. From the perspective of an author...the peer-review, is that as a peer-reviewer, you must never forget that there is another human who is ...thanks to our guest blogger Magdalena Julkowska from KAUST, Saudi Arabia. Magdalena Julkowska did her Ph.D....population in Amsterdam. Currently, she is a postdoc at KAUST, Saudi Arabia, in the group of Prof. Mark Tester...

...application is just as helpful as knowing when it will. And remember, antibodies are like puppies. Just because...bind to. Show specificity for the target antigen - Just because an antibody can bind to its target, does...suitability for an intended application - Remember, just because your antibody performs as expected in one...genes be easily knocked down or out.   Example: You just received an antibody that you want to use to look...transfected with your protein of interest and show just the faintest band from the cells transfected with...data that convinced you of your antibody’s trustworthiness. You may also consider sharing your validation...

.... In just a few weeks, we were able to connect them to a research group in Scotland, who had just the ...study has just been uploaded to the preprint repository BioRxiv. This example clearly illustrates the power... them are undergraduates or PhD students who are just starting their journey into science. Others are ...want to use this potential to make a difference not just against COVID-19, but also in other areas of scientific...

...large inserts can make lentivirus production frustrating - a researcher can complete an entire lentiviral...editing experiments. We aim to alleviate this frustration by producing, titering, testing, and providing...format. Finally, while we can’t currently produce custom gRNA-containing lentivirus, we are providing lentiviral...pooled gRNA library in plasmid format, a researcher must amplify the library, use next-generation sequencing...lentivirus format only deliver gRNA and therefore, must be used to infect a cell line already expressing...You need to make sure your assay is scalable and robust. What you don't need to do is spend your time troubleshooting...

...is to _________." Everyone in the room has likely just run in from some other commitment. An announcement...people in the room There are few experiences more frustrating than sitting through a meeting with no idea why...meeting at all. When no discussion is needed and you just need to give everyone a heads up (for example, notifying... where people can make comments. At meetings, we just focus on the parts where people had questions or...thing together. How long do you actually need? Just like liquids, meetings tend to fill the shape of... in-depth discussions can happen “off-line” with just one or two relevant people. How often do you need...

...not entirely. What truly unites us all is the frustration of an experiment failing because of a small, ...or your physical setup - to prevent the mistake, just like you would change variables to fix a failed ...longer need the full checklist. Great! But don’t just stop using it cold turkey. Step back gradually; ...an investment in accuracy.  Bench setup Small adjustments in the physical organization of your workspace...experiment in my head, double-checking details, or just taking a moment to relax. During really stressful...especially) suggestions you are initially resistant to. Just like any other experimental approach, use small,...

...alternative methods such as fluorescent microscopy must be used. To learn more about the most suitable fluorescent...membranes For intracellular protein targets, users must first fix proteins within the cells and then permeabilize...different populations of immune cells express unique Clusters of Differentiation cell surface markers (CD) these...up clumps before running on the instrument. To sustain cell health and limit debris, keep samples on ice...running a multicolor flow cytometry experiment, users must also prepare sets of controls to aid with compensation...dedicated detector. To ensure proper compensation you must include single-stained samples for each fluorochrome...fluorochrome in the experiment. Single-stained samples must be as bright or brighter than any experimental sample...

...Chemistry since 2001, making use of robust reactions that work in just about any situation (Devaraj & Finn...all of these situations. Specificity — coupling must happen in the right place in living systems when... lots of other biomolecules floating around that must not be touched. Click Biology Tags The broad functionality...Building Click Biology describes the search for robust, reliable reactions, using only nature’s building.... There are many convenient tools out there, not just relating to proteins but also covalent reactions...

... Breaks disulfide bonds HCl Adjust pH to 6.7–6.8 Adjust pH Pro tip! βME is a reducing ...membrane with antibodies on it if you plan to reprobe! Just as your sample protein is retained better, antibodies...you’re ready for round two (even if round two is just another attempt at the same target!). In the stripping...v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0–2.2 For the mildest stripping solution...times — some researchers report up to ten cycles! Just remember that a bit of sample is lost in each round...

... laser to the patch cable to the ferrule implant must be maintained during the entire protocol, otherwise...Laserglow, which caters to optogenetics users and adjusts the lasers appropriately (tuned for optogenetics...laser stimulation itself, the output of the laser must be controlled. As discussed above, each opsin variant... receptive to light once again. Therefore, light must be delivered in pulses. An example of a standard...are also available. Optimal stimulation parameters must also be determined empirically but it’s best to ...freely moving animals, the fiber optic patch cable must be the appropriate length for your specific behavioral...

...transferable skills guide: managing a team. You’ve just been promoted, congratulations! You’re now a manager...what needed to be done and how to prioritize but I  just added the “most important” tasks my schedule. This...should be questions! Remember that delegating is not just a way to create more time for yourself, but it will...and find a mentor Your manager should support you, just like you are supporting your report(s). This means...Make you team feel safe. Your team members need to trust you and you need to encourage them to speak their... What Makes a Good Mentor? Tips for Working in Industry and Success in Management Resources at Addgene.org...

...target type. To start the validation process, one must first confirm that a candidate antibody can detect...presence of supportive data. To be supportive, the data must show that the antigen being detected in the intended...chosen KO line are likely to express the target at a robust level, similar to that in samples of interest. ...amenable to genetic knockout, this represents a very robust approach to antibody validation. If your protein...difficult to achieve sufficient knockdown to enable robust assessment of antibody selectivity. Additionally...suppress the expression of many related proteins, not just your protein of interest, which can affect your ...ideally at endogenously expressed levels, and then adjust the approach to the specific end-use of an individual...

...their antibiotics   Ampicillin Ampicillin, often just called amp, is the historical king of the antibiotic... familiar?). Carb is degraded by beta-lactamase, just like amp is. Thus, the AmpR gene confers resistance...and eukaryotic selection, whereas other plasmids must carry both a bacterial and a mammalian antibiotic...="16" purpose="decorative" title="Check icon" %} Just bacteria {% icon icon_set="fontawesome-5.14.0" name...title="Check icon" %} Spectinomycin Excellent No Just bacteria Not SHuffle cells {% icon icon_set="fontawesome...

... roles by reaching out to my school district not just as a dad, but as a scientist. When our elementary...questions elementary students can ask. It doesn’t just need to be people who think of themselves as “scientists...on Twitter and got lots of suggestions. Here are just a few resources: Steam Powered Family  Biology...follows (which could be adapted for any topic--not just CRISPR): Who am I? Where do I work? The teachers...are interested in a career in education. Maybe you just like to see the look on a kid’s face when you’ve...like to thank the PTA of Butler Elementary for trusting me with Math & Science Night, and a special thanks...

...workflow management system As novices to creating a robust data pipeline that would be reliable and available...chose needed to fulfill certain requirements: Customizable to our specific needs Modern and well-supported...the order and usage of the scripts needs to be customized in the pipeline for the specific needs of the...Since each process can use a unique container, we customized each container for its particular purpose and... a complete pipeline however, required more than just identifying criteria that we needed. Prior to using...Nextflow script. With our completed pipeline, we could adjust the compute resources when needed, for faster runtime...