...which has been designed to perform gene transfer in vertebrates. The system is comprised of 2 components:... gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is expressed (red stars) ...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette... 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et al. Molecular evolution of a novel hyperactive...whose use is only likely to grow in the coming decades.  References 1. Kowarz E, et al. Optimized Sleeping...

...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses...delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H...Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here Additional...

...study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome?   The EBOV ...transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable of being replicated...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...as it would for a full-length viral genome (Figure 2). While it is technically possible to encode all four...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales...minigenome system Minigenome systems, such as the one described here, are extremely useful research tools. In ...

...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...choice in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...(4):251-66. doi: 10.1038/nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of...analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808. PMID: 22179551; ...

...Topic) 0:00 - 2:00 : Introduction to the podcast from producers Tyler Ford and Eddy Page. 2:00 - 3:55 : ...:56 - 7:04 : Alex describes the MD PhD process and explains why he loved the first 2 years of medical ...

...al. described the first light-activated tool for controlling neuronal activity, channelrhodopsin-2 (ChR2...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

... example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth review of quantitative... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ...interest from its production site to its final destination. As an example, most secreted proteins are produced...pathway to their subcellular or extracellular destination. At the end of their life cycle, proteins may...

... Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers. The...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals... CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...gRNA sequences without off-target effects for the 2+ million DHS sites. See below for further details....this article, Quilt CTO Aneesh Karve, shows how to design experiments that work anywhere in the genome. Aneesh's...enhancers! This leads us to a three-step approach for designing an experiment to identify the critical NANOG binding...

...Niroshi and Ben describe their projects 0:28 - 1:12 : Introduction from Tyler 1:13 - 2:02 : Niroshi and...and Ben introduce themselves 2:03 - 3:22 : How did you end up becoming a graduate student? 4:50 - 8:00...things get tough in the dreaded third year. Find Part 2 Here! We'd like to thank Niroshi and Ben for taking...

...question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1,...however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The protospacer flanking sequence...PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct RNase... and functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385...Cas13a as a diagnostic tool. A pool of DNA or RNA nucleotides containing a sequence of interest (red) is amplified...Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides are combined with Cas 13a in complex with crRNA...the target sequence is present in the pool of nucleotides, the nonspecific RNAse activity of Cas13a becomes...

...month, from 1/22/20–2/23/20, where there were zero plasmid requests. Afterwards, from 2/24/20–3/15/20 (the...Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received plasmid requests from ...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID-19 ...However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown in China...are no exception to this effect -- meticulously designed experiments in labs around the world were put ...requests correlate with paper publication The dataset includes 11,426 plasmid requests over 6 years for 35 papers...into two components: 𝜃x(t) and 𝜓z(t). 𝜃x(t) describes the exponential decay process we observed, while...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed... agarose gel. The resulting gel image includes a 1kb ladder (lane 1) that has bands ranging from about...

... TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2 Cerulean 433nm/ 475nm Lyn-tag (derived from the tyrosine-protein...based (chaining-based) cloning. The modules (Figure 2) are contained in individual plasmids and are each...between various cloning methods are listed in Table 2. For example,  chaining-based cloning methods require...suitable for combining endogenous sequences. Table 2: High Throughput Cloning Strategies Method Technology...PMID: 20205762. PubMed Central PMCID: PMC2822740. 2. Engler C, Kandzia R, Marillonnet S. A one pot, one...Origin and purpose of MXS chaining MXS-chaining was designed to create plasmids for fluorescence imaging or... can be assembled. For example, because CpG dinucleotides are underrepresented in most vertebrate genomes...

... systems to engineer better and faster ABEs (Fig. 2). These systems allow for rapid continuous protein... and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity..., Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR (2020) Phage-assisted evolution...

...scientists are using social media professionally, and 2) I needed to start using Twitter. Sharing Chemistry...but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn...PhD 👩🏾‍🔬💜 (@ThePurplePage) February 13, 2014 2. "The Scientist's Top 10 Guide to Twitter" blog post...you social media can positively impact your career, 2) given you tips for using various social media platforms...’s social media toolbox. Besides advice for using Twitter, this post provides suggestions for using LinkedIn...use Twitter as a scientist. While this post is designed with beginners in mind, there’s some great reminders...sheet from Science Media Savvy This tip sheet provides advice for tweeting and blogging as a scientist...

...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the... Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR labeling of...ortholog requires a different PAM sequence, a gRNA designed for one dCas9 should be specific to that ortholog...boxB Magenta boxB-MS2 White boxB-MS2-PP7 Despite the success of Ma et al.’s approach, fluorescently...genome editing. Such a system has been previously described for transcriptional activation/repression and ...

...both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best with insertions of 2 kb or... ivTRT to create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart. In the example ... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...using in mouse transgenics. Designing an Easi-CRISPR knock-in allele To design a knock-in allele at a given... PAM or guide mutation. Designing an Easi-CRISPR conditional allele The design for a floxed conditional...

...purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...cell suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...success was ~%50. If you get low knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells...PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari PS, Southgate B, Mccarten K, Monogarov G, ...knock-in. Another good design tool can be found at Desktop Genetics .For ssODN design, remember to incorporate...

... Multiple Introns.” Plant Communications, vol. 2, no. 2, Elsevier BV, Mar. 2021, p. 100135. Crossref, ...Constructs.” PLoS ONE, edited by Jean Peccoud, vol. 6, no. 2, Public Library of Science (PLoS), 18 Feb. 2011, p... vol. 5, no. 10, American Chemical Society (ACS), 2 May 2016, pp. 1059–69. Crossref, https://doi.org/10.1021...of Plant Genomes.” The Plant Genome, vol. 16, no. 2, Wiley, 9 Mar. 2023. Crossref, https://doi.org/10.1002...Crossref, https://doi.org/10.1186/s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex...Crossref, https://doi.org/10.1038/s41598-019-46171-2. Resources on Addgene.org Modular Cloning (MoClo...many MoClo kits are designed with specific model organisms in mind, others are designed for flexibility. ...

...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the different...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization through...characterization of the new variants is available here. Upgrades to iGluSnFR3 To improve upon the SF-iGluSnFR, ...