...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...be easily visualized.  The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...Rex's lab.  vfCRISPR, or very fast CRISPR, is a light-inducible CRISPR system developed by Taekjip Ha’...target DNA without cleavage until it's activated by light. Once activated, Cas9 cleavage can induce DSBs within...

...Although other methods have been able to achieve much higher activation, dCas9-VP64 is great for experiments...targeting single genes, SAM consistently shows the highest levels of gene activation compared to other CRISPR...Performance Generally, VPR is found to have significantly higher activation levels than the initial dCas9-VP64 activator...rearranging chromatin structure. This method has shown higher activation levels than SAM in some loci across ...has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou et al., 2018). ..., Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming...https://doi.org/10.1038/nmeth.3871 Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...

...current findings in nucleic acid science. I was delighted to see that the majority of attendees were scientists... enjoys preferential governmental policies for a high-tech development zone so many science and technology...fauna. I tried to find out what certain ingredients might be used for (like what would one take dried seahorse...blot results and we didn’t give tRNAs much thought. High throughput transcriptome sequencing methods including...families and provide a way to guide inheritance that might be less error prone than DNA replication. He too...things.” He further cautioned the distinction of “higher” and “lower” life forms as we shared millions of...the bacteria sensitive to known antibotics. These might be used on hospital surfaces and in hand sanitizers...

...daytime. They mostly stop emerging at night. An incubator with a light cycle makes for very predictable work...temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies develop faster but...When flies first emerge, their bodies will be very light in coloration and can look very plump. On the side...flies. Like Addgene, the Drosophila community is highly sharing-oriented, and there are many resources ...your fly gene using the search box in the upper right hand corner. They have video tutorials under the...useful information on topics such as choosing the right balancer chromosomes to use. If you’re not ready...

...should be easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting...near your insertion site, the corresponding gRNA might be undesirable due to potential off-target sites...they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9: NGT (4.5-fold efficiency...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly,...generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing efficiency over that ...Evolved Cas9 variants with broad PAM compatibility and high DNA specificity." Nature (2018) doi: 10.1038/nature26155...

...flow cytometry During conventional flow cytometry, light emitted by a fluorophore is guided to a single detector...on the other hand, utilizes the full spectrum of light to distinguish one fluorophore from another. Instead...upwards of 30-40 colors), spectral flow cytometry might be the better choice. Of note, there is no minimum...of the markers you are including in your panel. Highly expressed markers should be paired with dimmer ...markers with low expression should be paired with brighter dyes, so they can more easily be detected. Cytek...cells when designing your panel. If you have two highly overlapping cellular markers, it is important to...on the Addgene blog Antibodies 101:Selecting the Right AntibodyAntibodies 101: Secondary AntibodiesAntibodies...

...False, width='600', height='189', player_id='31668202421', style='text-align: right;' %} Figure 3. An...data with a 7-day rolling mean gave us a crude insight into the magnitude of the effect COVID-19 had on...there are many new COVID-19 cases being reported (right, red), likely due to quarantining {% video_player...most impacted or least impacted. These results highlight the instability and uncertainty in estimating ...methods for fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate...

...outperformed another CRISPR library with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries...CRISPRi sgRNA library While CRISPRko libraries are highly effective at identifying strong hits in genetic...because it's essential for viability. In addition, high copy number genes may mimic viability genes as they...knock-out library while mitigating toxicity induced by high copy number dsDNA cutting. A Dolcetto library containing...wide CRISPRko, CRISPRi, and CRISPRa libraries are highly effective for probing gene function while using... 1. Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation...

...a new coordinator for the annual Math & Science Night, I took the reigns. I also volunteered when our ... started coordinating the annual Math & Science Night, a chance for parents and other members of the community...activities. If your school doesn’t have a Math & Science Night (or some equivalent), here are some quick tips to...activities we had for my school’s Math & Science Night. There are plenty of places to get ideas for science...for all of these questions, and for any topic you might present to middle schoolers, is to provide current...again next year. There are many reasons why you might want to reach out to your school community as a ... Elementary for trusting me with Math & Science Night, and a special thanks to Joanne Marks, Jonathan ...

...Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work/life balance. Is it truly possible to...have one-half of the guilt. You can handle that, right? Big tip: this is an important topic to discuss .... The same with sheets – wash them and put them right back on the bed to save a step! There is more than...situation. Go to church, join a club, meet the neighbors and create a network of support to help with elder... elder care, carpooling, cooking on nights when you can’t get home, babysitting or running errands. Give...very, very fast! I will never forget the time my eight-year-old daughter vehemently reminded her doctor...wait and some things can’t. I kiss my kids good night every evening I am home no matter how busy I am....

...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today: Career Planning Resources...graduate school and beyond is to live in your lab, right? Wrong! While it is clearly a good idea to be diligent...there are different types of conferences that you might attend. Societies, such as the American Society ...contact an email with the relevant info and they just might personally hand your credentials to HR, greatly ... institution’s career center. Alternatively, it might even be a colleague. A mentor does not necessarily...

...from magnetic beads are equivalent, if not slightly higher, than those of agarose beads, despite the size... method is appropriate for most situations.  A slightly different method is desirable if the antibody ... crystallizable region (Fc) of antibodies with a high affinity (Hjelm, 1972, Bjorck 1984). Protein A, ...beads, break weak protein interactions, and limit high throughput procedures. However, agarose beads are...the protein of interest. The wide availability and high specificity of anti-epitope antibodies make this...Tagged proteins are typically expressed at much higher levels than endogenous proteins. Data obtained ...

...such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing efficiencies...Every few months, we highlight a subset of the new plasmids, antibodies, and viral preps in the repository...the utility of this technique.  By engineering a light-sensitive LOV domain into the TurboID enzyme, Lee...LOV-Turbo becomes active when illuminated with blue light, catalyzing the biotinylation of nearby proteins...Cheah, J.S., et al. (2023). Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal...

...Central PMCID: PMC3150193. 8.Han, Xue, et al. "A high-light sensitivity optical neural silencer: development...in your brain region of interest could be low or highly variable without the behavioral test. Another readout...important in memory, an appropriate behavioral test might be contextual fear conditioning (reviewed in [4]...optogenetics setup (viral expression, efficiency of laser light transmission to the target neurons, appropriate ...mistakes. The easiest way to learn optogenetics is straight through the source - find a lab within your own...Filippo, and Claire Wyart. "Optogenetics: a new enlightenment age for zebrafish neurobiology." Developmental...

...need an expression strain? Protein expression from high-copy number plasmids and powerful promoters will...typically expressed in E. coli engineered to accomodate high protein loads using inducible promoter systems (...to all or most expression strains to accommodate high protein levels including:  ompT: Strains harboring... (DE3) pLysE* Chloramphenicol (pLysE) pLysE has higher T7 lysozyme expression than pLysS; expression vector... (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin reductase and glutathione reductase...—the difference being that the latter provides tighter control of basal expression. What if I don't see...media such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media...

...canonical base editing window, base editing has higher efficiency and fewer indels than prime editing....However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning...varies widely — it still almost always results in higher ratios of desired edits to indel byproducts than...average 2.3- to 5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1 ...process of double nicking increases indel formation slightly. Designing the sgRNA with a spacer that only binds...asked. Hopefully this table helps you keep them straight! Innovation Purpose PE2 PE3 PE4 ...PE6 PE7 Pentamutant reverse transcriptase Higher efficiency ✓ ✓ ✓ ✓ ✓   ✓ Second...

...institutions of higher education nonprofit entities related to or affiliated with institutions of higher education...reserved for individuals with a US Master’s degree or higher degree. These numerically limited visas are referred...theoretical and practical application of a body of highly specialized knowledge in a field of human endeavor...the cap if the applicant works at institutions of higher education, a related or affiliated nonprofit entity...interest waiver criteria however the EB-1A requires a higher standard of proving that you are one of the top...showcases (not usually applicable to scientists). High Salary –not relevant to most postdocs. Commercial...circulation) in the field EB-1Bs historically have a high rate of approvals with USCIS due to the fact that...

... In this quarterly blog series, we’ll highlight a few of the new CRISPR plasmids available at Addgene...recently developed a platform called SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) to detect... this further to provide multiplexed detection, higher sensitivity, and more. Read the paper in Science...developed dual-nuclease Cas9-Cas9 chimeras with higher target site activity and more predictable and precise...inhibitor, with the LOV2 photosensor from Avena sativa, light-mediated genome editing is now possible. Learn ...

...Proximity labelling relies on the labelling of neighboring proteins within a cell with biotin. Biotin, is...avidin coated purification techniques. Due to the high affinity of biotin for avidin, proximity labelling...promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin based proximity labelling...from the ligase and bind to exposed lysines on neighboring proteins. Thus to capture protein complexes, ...electron-rich amino acids such as tyrosine on neighboring proteins resulting in their biotinylation. The...The advantage of APEX is its speed, labeling neighboring proteins in minutes rather than hours like BioID...sensitivity. Sensitivity could be increased with higher expression levels but for many fusion proteins ...

...some of these plastics are high grade and can be washed and reused. This might sound risky in terms of contamination... in a high-throughput and efficient way? Grenova, for example, offers automated, benchtop, high throughput...are an accepted part of lab culture, but with the right incentives (for example, by grant agencies), we ...behavior and innovate solutions and practices to meet higher environmental standards. Find some easy action ...