...Escherichia coli and Agrobacterium tumefaciens. 3 Biotech, 13(3), 91. https://doi.org/10.1007/s13205-023-03507...plasmid RK2. Molecular & General Genetics: MGG, 152(3), 129–135. https://doi.org/10.1007/BF00268809  Prior...plasmids may have high copy numbers and reduced metabolic load (Meyer 2009). Figure 1: Idealized ...

...at the 5′ or 3′ end of the sgRNA (i.e. TOP1 or TOP2 constructs; see Supplementary Note 3 in the article...Polstein LR & Gersbach CA, Nat Chem Biol 2015 Mar;11(3):198-200. SunTag system for single molecule imaging...protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein ..., (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous expression of cDNAs and shRNAs with ...8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various pieces for inducible... 2012, Adam Cohen's lab presented Archaerhodopsin 3 (Arch) as a new rhodopsin based voltage indicator ...shorten the half-life of both the luciferase mRNA (3’UTR AUUUA [5 consecutive]) and protein (C-terminal...

...without investing time and labour on reagent preparation in the lab. Figure 3: csRNP-assisted epitope tagging...-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first...we could tag >60 transcription factors in a short (3-week) time window; this was possible because reagents...PMID: 29638216. PubMed Central PMCID: PMC5947990. 3. Jarvik JW, Telmer CA. 1998. Epitope tagging. Annu...ChIP-seq methods provide a wealth of information about how protein networks work in cells. A major bottleneck...two-part cr/tracrRNAs for CRISPR experiments (in collaboration with Integrated DNA Technologies, USA). These...first crRNA we designed for ~250 genes, we have had about 30% tagging success, although many of these genes...

...to supply it with another plasmid.   Figure 3: Comparison of Multiplex Strategies including Standard...For example, if you were to develop an array using 3 distinct spacer-repeats, you could easily create 7...a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...PMC4231726. Find plasmids from this paper at Addgene. 3. Sakuma, Tetsushi, et al. “Multiplex genome engineering...multiplexing options, but also check out our blog post about multiplexing with Cpf1.   Figure 1: Multiplexing...assembly.  Unlike some of the plasmids described above, this vector does not contain Cas9, so you’ll need...similar to the Csy4 polycistronic system described above, except that they use an endogenous nuclease system...

...spring of 2015 and since that time have received about 3 project proposals per month.  We are currently ...greater chances for success.  Build a team of 2 or 3 Don’t go it alone.  You’ll find that no matter how...

...students who are passionate about communicating science.  ComSciCon's events have 3 main goals: To help graduate...succeeded in communicating the information you intended. 3. Join the scicomm community Like most aspects of science...  If someone asks a question about something they did not understand about your presentation, question ...important and relevant to them.   When you think about it, virtually every milestone in a scientist's career...scientists learn from advisers and other mentors by collaborating as they craft sentences for abstracts and presentation...  If someone suggests a change to a slide, think about whether the same principle applies to other slides...much bigger job than merely telling your audience about the work you've done. If you want to maximize the...

...exchange, the government protects (and often funds [3]) scientists as well as other citizens. Thus civilization...depending on the number of people supporting it.  3) Linking scientists to one particular political party...request of this administration promises more cuts (3, 12, 13, 27). In order to achieve the breakthroughs... with a testable hypothesis - an educated guess about how some part of the world functions. Experiments...governments exist in reality and science is how we learn about reality. Formally, a government can be defined ... More concretely, scientists gather information about reality informing people and their governments on...people to rally for science, others are concerned about possible unintended consequences of a march. Three...

...some impressive videos of these interactions (Ref 3). You can find useful tips on in vivo imaging in our...Research 9.12 (2016): 895-905. PubMed PMID: 27913448. 3. Cai, En, et al. "Visualizing dynamic microvillar ...Cancer, Inflammation and Immunity and learn more about “... recent findings in basic immunology, cancer-immune...curing cancer in cross-disciplinary projects and collaborations. Summarized by Professor Michael Karin from...This was followed by a talk from Professor Karin about alcohol and obesity as driving factors for chronic...got a chance to talk with Dr. Friedmann-Morvinski about tools used to analyse the tumor microenvironment...community, contact [email protected] to find out more about our free deposit and distribution service! References...

...-lantern emitted yellow-green light and exhibited 3-5 fold increased brightness over previous BRET probes... single-cell and whole-body imaging.” Nat Commun. 3 (2012): 1262. PMID: 23232392 PMCID: PMC3535334 Find...Since their first publication in 2012, the Nagai laboratory has assembled a collection of multicolored nano-lanterns...Nano-lanterns, Takai et al. continuously imaged cells for about 4 hours! The latest enhanced Nano-lanterns (eNL)... Nano-lanterns and your experiments The Nagai laboratory has a done a wonderful job characterizing Nano-lanterns...Additional Resources on the Addgene Blog Learn about Photoactivatable Fluorescent Proteins Check Out ...

...chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-...antibiotic selection here at Plasmids 101, we can talk about an even more specific method of screening your cloning... plasmid is a great start when cloning, but how about being able to choose those that contain plasmid ... a widely used method to do just that! (To read about other screening strategies, check out our screening... called α-complementation. The system described above was put to practical use in the following way. Scientists...

...abundant incorporation of cysteine in their proteomes (3). This led to the eventual discovery of a naturally...which can be specifically recognized by the Sep-tRNA. 3. An expression plasmid encoding the ORF to be expressed...PMID: 25514926. PubMed Central PMCID: PMC4383998. 3. Li, Tong, et al. "Cysteinyl‐tRNA formation: the last... one of the MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic...non-phospho species on PhosTag gels as described above. Reagents and protocols Sep-OTSλ plasmid - Addgene...interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion. You can follow...Blog Verify Your Constructs with Colony PCR Read about Tags for Protein Purification Resources on Addgene.org...

...tumor-infiltrating lymphocytes of that tumor. In Figure 3 you can see how I gated out my lymphocyte population...look for them on the FSC-A/SSC-A plot. Figure 3: (A) With the acquisition voltage (not shown) used...gates while some like to make looser ones (Figure 3). A more “liberal” gate in the beginning should not...lymphocytes in a mouse tumor sample You made it! So much about the theoretical background. Let’s dive into the ...often choose a looser gate here. I am not worried about including irrelevant events in my analysis, as these...population, acquired from the previous gating pipeline, about 53% of my cells are dendritic cells and ~13% are...ve covered in this blog post. First, you learned about the types of scattering that occur when a cell is...

...incorporation of a 3’-5’ exonuclease in the reaction mixture. The exonuclease removes mismatched 3’ overhangs,...scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check Out Our Plasmids 101 eBook! ...to produce sgRNA for CRISPR experiments can be laborious, requiring multiple cloning steps. You can simplify...Additional Resources on the Addgene Blog Learn More about Gateway Cloning Gibson Assembly and Other Long-Homology...

...used saving the researcher both time and resources (3). Still, researchers generating transgenic animals..., called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl group required for the extension of a DNA...Medicine 307.1 (1982): 30-32. PubMed PMID: 6176866. 3. Hoebeeck, Jasmien, Frank Speleman, and Jo Vandesompele...abundant in a sample. Whereas Southern blotting is labor intensive and requires a large amount of high-quality...DNA technologies that are now commonplace in laboratories. As scientific technologies rapidly progress...PMC4411503. Resources on the Addgene Blog Learn More about Restriction Cloning Get Tips on Analyzing DNA Sequencing...

...Figure 3 shows a histogram for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example...interest. Created with biorender.com. The plot above is for a smeary expression and is quite different.... Additionally, you can gate off the histograms above, allowing you to visualize your data as a dot plot...

...the core of a Shine-Delgarno sequence found at the 3’ end of the leader sequence. Using a variety of GFP...create custom RNA binding proteins in as little as 3 days. Abil et al., 2014 show that you can fuse custom...customized sequence specificities." Nature communications 3 (2012): 1147. Cooke, Amy, et al. "Targeted translational...PUF Assembly Kit Below you’ll find information about what these kits contain, how they can be used, and...encoded sensors enable real-time observation of metabolite production." Proceedings of the National Academy...

...stable gRNA than when they inserted the aptamer at the 3’ end of the gRNA. Because of the success of the MS2...can be increased from 2-fold (by CRISPRainbow) to 3-fold with CRISPR-Sirius. Triple color detection With...the Addgene blog Download our CRISPR eBook Learn about fluorescent protein-based tools Read all CRISPR ...

...Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire, G. Roman, R. L. Davis, Gene expression...Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z... use the Gal4/UAS system, and you can read more about them in review articles (2–4). You can also take...

...resulted in a roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially...PMID: 18500252. PubMed Central PMCID: PMC2574934. 3. Zingg, Brian, et al. "AAV-mediated anterograde transsynaptic...diluting - if you end up concentrating your AAV above your desired titer and want to dilute it, err on...

...repression, high precision edits, and more. Figure 3: Schematic of the basic CRISPR-Cas9 mechanism, representing...double-stranded breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency...for genome-editing applications. Molecular Cell, 84(3), 463-475.e5. https://doi.org/10.1016/j.molcel.2023.12.034...the type I CRISPR system. Gene and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013...Class 1 is the most abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly...CRISPR systems called CasPEDIA, with information about enzyme activity, experimental conditions, and more...