...normal size.      References: 1. Doench, John G., et al. "Optimized sgRNA design to maximize activity and ... producing a wall of results that can only be described as bigly. But such a project should not be undertaken...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using CRISPR...with CRISPR/Cas9 Get John Doench's Tips on gRNA Design Check Out News Ways to Use CRISPR for Single Base...

...organized and boost your productivity in 2014. Tip 1: Use color coding, labels, and post-its to make finding...lab earlier, to read that stack of papers on your desk, and to tackle some new experiments before the next...gathering that pile of lab notebooks and binder on your desk. Arrange them in chronological order (or by project...labels. Color code your Post-its and write brief descriptions on each. Place the notes sticking up out of ...colleagues. Start small by identifying the protocols, guides, or references that your labmates ask to borrow...

...base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with...or one pyrimidine base to the other. Figure 1: Base transition editors catalyze purine-to-purine...engineering and protein delivery. Nature Communications, 8(1), 15790. https://doi.org/10.1038/ncomms15790 Richter...DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s...named ABEmax. In 2020, two papers were published describing additional ABEs evolved from ABE7.10 that have...cytosine and adenine base editing, called CABE-Ts and TadDEs without additional fused components (Lam et al....

...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system: a vector...subtilis and Pichia pastoris pCri also includes plasmids designed for protein expression in systems other...construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed...plasmids are divided into two sets, each of which includes plasmids with different varieties of tags, both...other eukaryotes better than E. coli. Plasmids designed for these systems may include a signal peptide...can improve disulfide bond formation. pCri also includes a special feature for difficult-to-purify membrane...membrane proteins. pCri vector pCri-13a, designed for use in B. subtilis, contains the MISTIC (membrane-...

...target cell line or organism (Presented in figure 1). More information on Agrobacterium-mediated transformation...mediated cloning of your gRNA of interest. Figure 1: Simplified schematic of agrobacterium-mediated transformation... While it is certainly possible to select for desirable traits using traditional plant breeding approaches...modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already been used...wheat, mushroom, and rice (Khatodia et al., 2016). Despite the almost universal functionality of the CRISPR...genes in plants using the same general experimental design principles developed in other model organisms (...nanostructures (Zhang et al., 2019) or cell-penetrating peptides (Santana et al., 2020) and plants viruses (barley...

...products by their environmental impact on a scale of 1-10: the lower the score, the lower the environmental...collect in those red biohazard bags, typically is destined for a landfill. However, Triumvirate Environmental...Then, they use this material to produce various grades of plastic lumber and distribute it to industrial...

...deletions, and all possible base-to-base changes.   1. Plasmids 101: Transformation, Transduction, Bacterial...on the Addgene blog and have created and updated guides on various topics in biology over the past year...containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for the Academic Job Interview...

...resume that will give you a chance at an interview:  1) Have an excellent resume ready to be submitted The...experience. Most Addgene PhD level job descriptions clearly describe that the roles do not involve bench ...submission of a job application. 3) Read job descriptions carefully Be open minded and be bold. You don...qualifications to apply for a position.  A job description is usually a wish list and the hiring manager... way to a hiring manager. Learn to confidently describe your strengths in your resume and don't be shy...

...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...databases, DNA sequence manipulation, and primer design. Best wishes for the new year and your research...

...to western blotting  In a western, proteins are: (1) separated by size, (2) transferred to a membrane,...  (3) detected using antibodies.   Figure 1: Overview of the Western blot process. The sample,...protein analyses using ELISA. Peptides 72:4–15 . https://doi.org/10.1016/j.peptides.2015.04.012 Burnette WN ... Figure 2: The western blot dry apparatus includes a stack of filter paper, a copper anode/iblot ...blocking buffers, and incubation times. If the vendor includes publications that cited the antibody, consider...abundance In addition, the thickness of the band provides information about the relative abundance of the...

...2013 edition. 10 new developments in open science 1)      In February, the Office of Science and Technology...     Science’s John Bohannon published an article describing a sting operation in which he submitted a phony...

...science.aaq0180  Fine EJ, Appleton CM, White DE, Brown MT, Deshmukh H, Kemp ML, Bao G (2015) Trans-spliced ...indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically...as potent and specific? The two AAV strategies described above showed successful target modification, indicating...from Streptococcus thermophilus and a rationally-designed truncated Cas9. Unfortunately, certain drawbacks... approximately 1350 amino acids in size, which includes SpCas9, and shortern orthologs approximately 1000...Split inteins are a pair of naturally occuring polypeptides that, when at the ends of two proteins, mediates... proof-of-concept split intein SpCas9 which had modest editing rates in HEK-293T cells when compared to...

...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse of...cellular analysis products at Promega.     References 1. Hall, M.P., et al. (2012) Engineered luciferase reporter...above 600 nm. LumiFluors Schaub, F.X., et al. (3) designed two imaging probes. One consists of NLuc fused...Reporters. The LumiFluor reporter constructs were designed to be packaged into a retrovirus for infection...BRET probe for imaging. These authors set out to design a better fluorescent protein that could be co-imaged...acceptor, the more powerful the FRET signal. In well-designed FRET-based biosensors, the intensity of the FRET...

...relations, advocacy, and writing legislation (see Figure 1 for career map). The beautiful part of science policy...about science policy as a profession. Figure 1: Science policy can lead you to many paths! For a ...uncertainty of it all. Though I had spent the first few decades of my life following a very straightforward conveyor... harassment in academia or a large company who decides how they are going pursue regulatory approval for...their Local Engagement Playbook for step-by-step guides on how to engage in your local community.  Read...comprehensive resource developed by Dr. Kelly Singel which provides an overview of all available science policy fellowships...

..., or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent tags in S. cerevisiae, ...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...integrated. Simply design your amplification primers with the desired targeting homology—in frame, of ...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

...depends on what you are looking to study. Figure 1: A schematic showing the differences in chemogenetics... Roth BL (2015) DREADDs (Designer Receptors Exclusively Activated by Designer Drugs): Chemogenetic Tools...less invasive and more flexible as it relies on designer ligands that can be simply introduced by injection...lab and Takafumi Minamimoto’s lab known as deschloroclozapine (DCZ) has been developed to meet these requirements...should be carefully controlled for in experimental design.   Optogenetics Chemogenetics Timing Fast ...Find our chemogenetics and optogenetics science guides ...

...information Since the inception of Phage Directory ~1 year ago, Sacher and Zheng found that they could expand...infection, she shared that tweet with Jan Zheng, a UX designer that she knew. “We had actually met at a lindy...to possess the correct phages will test. These includes labs with a large number of different phages or...

...Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle M., et al...wall of green algae (Soo et al., 2015). There, it divides into two cells. At the same time, it sucks out ...1983). Grasshopper parasite The parasitic worm Spinochordodes tellinii‘s larvae makes its home inside grasshoppers...

...problem - see our recap below! Day 1: gRNA design tools: How to Design your gRNA for CRISPR Genome Editing...given our CRISPR Plasmids and Resources a fresh new design, sorting the plasmids we have available by category...capabilities. Epigenetics: CRISPR has made it possible to design programmable epigenome editing tools, and the field...cleavage in bacteria. In mammalian systems, Cas13a degrades only the specified RNA target. Bonus: It displays...

...  Figure 1: Overview of the parts of CRISPR. The bacterial chromosome encodes a tracrRNA (in some...DNA sequences. In laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “single-... sequences in the CRISPR array that will become guides for future targeted degradation. Before integration...biology of Cas9. Want a simpler guide to keep at your desk? We’ve got a CRISPR cheat sheet for that! Resources...