...cutting results in 5’ four base overhangs complementary to the overhangs of the assembled inserts. After...in an orientation that results in 5’ four base overhangs after cutting. The destination vector receives... backgrounds – so much so that there was little range for detectable improvements in the methodology. ...Gate assembly, ligation of mismatched pairs of overhangs can lead to incorrect assemblies, so care must... ligation by this enzyme for all 3- and 4-base overhang sequences under standard reaction conditions. ...identification of mismatch-prone pairings. Using these 4 bp overhang observations, accurate “high-fidelity” junction...can be applied to derive similar high-fidelity overhangs for any Golden Gate assembly design. Number...

... The biggest change is to our most frequently used feature: the search bar. We’ve changed the colors and...Starting today, you may notice some changes to our website! Part of sharing science and making access... development, and content teams have made some changes to the look and feel of addgene.org. Some you’ll...away, while others may be less obvious — but every change is meant to make your experience on our website... on “Deposit Materials” and the search bar will change to an easy-to-use depositing guide, with links ...antibody of the day While those are the biggest changes, there are plenty of less obvious ones to discover...

...thanks to our guest blogger, Chinmaya Sandangi! Dr. Chinmaya Sadangi is currently a postdoc at the University...post was contributed by guest blogger Chinmaya Sadangi, a postdoc at the University of Toronto. The Addictive...communicating science to non-scientists. Chinmaya Sadangi, a postdoc at the University of Toronto, created...underrepresented minorities to study STEM subjects. Here, Sadangi and his team answer a few questions about The Addictive...while “manipulate” could mean “worked with” or “changed” to scientists. Keep your message to the main three...Germany in 2017. Apart from The Addictive Brain, Dr. Sadangi is an eLife ambassador, SfN community leader, and...

...to iPSCs and re-establishment after Partial rearrangement from one cell type to another Time and resources...vitro as part of the process. This opens up a wide range of possibilities in clinical situations. In cases...19998274. PubMed Central PMCID: PMC2946078. 16. Zhang, D., et al., Highly efficient differentiation of...21151107. PubMed Central PMCID: PMC3033971. 18. Huang, S.X., et al., Efficient generation of lung and ...21434814. PubMed Central PMCID: PMC3161101. 20. Chang, C.J., et al., Production of embryonic and fetal-like...2010. 5(1): p. e8763. PubMed PMID: 20098701. 31. Tang, Z.H., et al., Genetic Correction of Induced Pluripotent...PMCID: PMC4835250. 32. Ma, L., Y. Liu, and S.C. Zhang, Directed differentiation of dopamine neurons from...

...PMC2629495. Zingg, B., Chou, X., Zhang, Z., Mesik, L., Liang, F., Tao, H.W., and Zhang, L.I. (2017). AAV-mediated...24694006. PubMed Central PMCID: PMC4137353. Chan, K.Y., Jang, M.J., Yoo, B.B., Greenbaum, A., Ravi, N., Wu, W...PubMed PMID: 18223548. Howard, D.B., Powers, K., Wang, Y., and Harvey, B.K. (2008). Tropism and toxicity....A., Ng, L., Winslow, B., Cain, N., Mihalas, S., Wang, Q., Lau, C., Kuan, L., Henry, A.M., Mortrud, M....Wohnoutka, P., Gerfen, C.R., Koch, C., Bernard, A., Dang, C., Jones, A.R., and Zeng, H. (2014). A mesoscale...Hisey, E., Tanaka, M., Kearney, M., Chattree, G., Yang, C. F., Shah, N.M., and Mooney, R. (2017). Identification... 195–206. PubMed PMID: 17343566. Tervo, D.G.R., Huang, B.-Y., Viswanathan, S., Gaj, T., Lavzin, M., Ritola...

..., Villiger, L., Zhou, W., Jiang, K., Garushyants, S. K., Roberts, N., Zhang, L., Vakulskas, C. A., Walker...base-to-base conversion in principle, but some changes require multiple steps and enzymes. Prime editing...can encode any of the 12 possible base-to-base changes in a single edit. Prime editing is also more precise...breaks has been widely used to generate precise changes. However, the efficiency of Cas9 cleavage is relatively...al., 2023). Therefore, they decided to evolve a range of specialized prime editors (PEs). PE6a and PE6b...lab! This post was originally written by Jennifer Tsang in October 2019 and updated by Emily P. Bentley ...Chen, W., Suiter, C. C., Lee, C., Chardon, F. M., Yang, W., Leith, A., Daza, R. M., Martin, B., & Shendure...

... map the turnover of Langerhans cells in the dermis and identify that Langerhans cells are replenished...membrane-tethered CFP (cyan). Brainbow 1.1 adds OFP (orange) (Livet et al., 2007). Brainbow uses a Cre/lox ...respectively, cells will fluoresce either red or orange until recombination occurs. Following the development...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically... to develop a system for tracking the fate of Langerhans cells. In the Ubow mouse, cells expressing YFP...through the proliferation of specific, and not all, Langerhans cells, to create foci of proliferating cells...., Clément, et al. "Multicolor fate mapping of Langerhans cell homeostasis." Journal of Experimental Medicine...

...the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar range. For more details... milliliter can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath...powered by a 9V battery which uses a Blue LED and an orange filter to detect fluorescence upon cleavage. Highlights...the immobilised target sequences. Upon binding, changes in fluorescence can be visualised. Another approach...nucleotide variations. Also, FnCas9 cleaves in a range of temperatures varying between 10-50°C. Visit Addgene's...Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F (2019) SHERLOCK: nucleic acid detection with CRISPR...Laboratory. https://doi.org/10.1101/2020.04.20.052159 Zhang, F., Abudayyeh, O. O. & Gootenberg, J. S. A protocol...

..., 343-345. PubMed PMID: 19363495. 2. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR... the 5’ ends of the fragment, generating long overhangs which allows the single stranded regions with ...described in a recent publication by the Lockey lab (Wang, et al. 2015). In this case, rather than using a... along with CRISPR was also described recently (Jiang et al. 2015), in which very large fragments of the... it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, Zhu TF...

...with all these questions, Dr. Le Cong and Dr. Feng Zhang developed a Google Forum to allow scientists to ...more Q&As which focus on genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR...Double nickase' is a new system, developed by the Zhang lab, which has comparable efficiency to the optimized...nickase system, including one recently from the Zhang group. Learn more here. Q2: When designing oligos...without the G. Because the G is only one base, the Zhang lab usually adds it when they order the oligo. If...them up, then perform individual genotyping (using Sanger sequencing, for example) on each colony in order...purification by performing a genotyping assay (such as Sanger sequencing). Click here for a useful reference...

...Mi-Yoon Chang, Baek-Soo Han, Sanghyeok Ko, Eungi Yang, Kwang Yul Cha, Robert Lanza, and Kwang-Soo Kim.... 2677165. 3. Jia, Fangjun, Kitchener D. Wilson, Ning Sun, Deepak M. Gupta, Mei Huang, Zongjin Li, Nicholas...includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm independent...using EBNA1 based vectors is that the plasmid can hang around for awhile, even after selection is removed...

...enzyme, or enzymes that have compatible overhangs or no overhangs after digestion, you will need to use ...with restriction enzymes to generate compatible overhangs that allow them to bind. Ligase is used to make...ended up in the direction you want - usually by Sanger sequencing or further restriction digests. Of course...inserts cut out of it and your new insert from any overhangs cut off of it via their different migration speeds...insert in a single tube allowing the compatible overhangs generated by restriction digestion to anneal to...only one enzyme or used enzymes with compatible overhangs for your ligation, then you will need to verify...appropriately sized insert, you should send it for sanger sequencing using primers that will allow you to...

...Figure 2: The steps of colony PCR.   Sanger sequencing Sanger sequencing determines the precise order... you'll want to design a custom primer. Because Sanger sequencing can usually sequence just 1 kb of DNA...post for tips for analyzing and troubleshooting Sanger sequencing results. At Addgene, we now use next-generation...that NGS verification is more time-intensive than Sanger sequencing or the other techniques mentioned above...Learn more about Sanger sequencing Read the Molecular Biology Reference ... Jennifer Tsang...

...fluorescent output, which will turn on, change color, or change intensity of fluorescence in the presence...receptor, but readouts could involve fluorescence, pH change, heat, etc. The last component of the biosensor...engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way to acetylcholine...fluorescence-based and have been optimized for a wide-range of uses to monitor the fundamental cellular building...actinin will produce different signals based on changes to the structure of actinin in low vs. high tension...real-time cellular reactions to stimuli and as well as changes in cellular dynamics during so-called housekeeping...

...CRISPR technology has changed how scientists edit and control genes, but according to the Broad Institute's...December, Konermann and her colleagues in Feng Zhang’s lab describe just such a system, which they call...perfect complement for one conducted earlier by the Zhang lab for GeCKO, a lentiviral CRISPR library enabling...enabling genome-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here we have shown ...in the genome for gene control over a 1,000-fold range. Weissman explained that there are two levels of... Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Cell. 2014 Feb 27;156(5):935-49. doi... Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Nature. 2015 Jan 29;517(7536):583-8. doi: 10.1038...

...through filtration methods because of its ability to change shape and the absence of a rigid cell wall. Here.... For testing, the medium should not have been changed for at least two or three days before sampling ...commonly infecting mycoplasmas are used. A broad range of primers encompasses almost all the infecting ...chemical reactions and this induces a noticeable color change. This method has high sensitivity but low specificity...are working without gloves or masks. Make sure to change the gloves and masks regularly instead of keeping...water provided to maintain the humidity should be changed regularly as mycoplasma, bacteria, and fungus can...contamination, easy to use, and effective for wide range of mycoplasma. These methods cannot guarantee complete...

...transformed the palette of experiments possible in a wide range of organisms and cell lines. In C. elegans, one ...at 25 ºC. This method resulted in efficiencies ranging from 11-100% of F1 animals carrying precise knock-ins...This stringent selection allowed me to optimize a range of editing parameters: oligo repair templates with...injected animal.  PAM sequence considerations For changes 5’ to the PAM, sequences corresponding to the same...locus (CRISPR-ChAP-MS) could further transform the range of experiments imaginable in C. elegans. Equally... and systems, allowing rapid progress in a wide range of fields.   Thank you to our guest blogger! Jordan...//doi.org/10.1534/genetics.118.301532 Ebbing A, Shang P, Geijsen N, Korswagen HC (2017) Extending the ...

...set-up has been changed (for instance after replacing a broken lightbulb or exchanging excitation filters...fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017) and photoswitching...fluorescence signal, showing no or little deterioration or change of the signal during the course of the experiment...and by comparing how the fluorescence intensity changes over time, the photostability of the fluorescent...comparable (lower panel of figure 1). To avoid changes in excitation power, it is important to perform...filters). This avoids substantial changes in excitation power and will help to keep the photostability of...

...in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined the function ...completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out experiments are .... doi:10.3390/ncrna7040079 Horlbeck MA, Liu SJ, Chang HY, Lim DA, Weissman JS. Fitness effects of CRISPR...(5):573. doi:10.1038/s41587-020-0428-0 Rinn JL, Chang HY. Genome Regulation by Long Noncoding RNAs. Annual...10.1146/annurev-biochem-051410-092902 Liu Y, Cao Z, Wang Y, et al. Genome-wide screening for functional long...Nature. 2016;539(7629):452. doi:10.1038/nature20149 Jiang Q, Meng X, Meng L, et al. Small indels induced by...2015;6. doi:10.3389/fpls.2015.01001 Ho TT, Zhou N, Huang J, et al. Targeting non-coding RNAs with the CRISPR...

... By Jennifer Tsang GFP-based Touching Nexus (G-baToN) is a new tool developed by Rui Tang in Monte Winslow...the sender cell and the receiver cell. Image from Tang et al., 2020.   G-baToN can also be applied ...modulating the interactions between cells.      Tang et al., eLife 2020. https://doi.org/10.7554/eLife...conditionally degrade proteins of interest in cells (Zhang et al., 2015). Recently the Ward lab created a tool-kit...genetics/iyab006       The CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids...of Cas enzymes. CRISPR-ARE is a new system from Jiazhang Lian’s lab that enables simultaneous gene activation...