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TypeBlog Post...reaction. The destination vector and entry vector(s) are placed in a single tube containing the Type IIS...silent point mutations at internal recognition site(s) thus eliminating these from your gene of interest...throughput capability. Engler C, Kandzia R, Marillonnet S. PLoS One 2008;3(11):e3647. doi: 10.1371/journal.pone...PubMed. Golden Gate cloning. Engler C, Marillonnet S. Methods Mol Biol. 2014;1116:119-31. doi: 10.1007/...
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Reflections and Looking Ahead
TypeBlog Post...Trimmer, J. S., Rego, M., Freedman, L. P., Ferrara, F., Bradbury, A. R. M., Cable, H., & Longworth, S. (2024...References Ayoubi, R., Ryan, J., Gonzalez Bolivar, S., Alende, C., Ruiz Moleon, V., Fotouhi, M., Alqazzaz... -
Mapping the 4D nucleome with CRISPR/Cas9
TypeBlog Post...Reyes-Gutierrez P, Wolfe SA, Zhang S, Pederson T. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7. doi...FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make this... -
Zinc Finger Consortium: OPEN Reagents
TypeCollection... zinc-finger arrays. Maeder ML, Thibodeau-Beganny S, Sander JD, Voytas DF, Joung JK. Nature Protocols ... -
Empty Backbones - Choosing Your Perfect Plasmid Backbone
TypeCollection...expression empty vector pFA6a vectors - Epitope tagging in S. pombe Insect/Baculovirus UAS, MT, Polyhedrin Targeted...expression pFA6a vectors - PCR-based C-terminal tagging in S. cerevisiae pCS2FLAG - N- or C-terminal ...display of proteins on the extracellular surface of S. cerevisiae cells His-tagged versions of pFastBac ... Plasmids page pBF3060 - Metabolic engineering in S. cerevisiae TALENs Gene targeting TALEN kits - Construct...capture and expression of bacterial gene clusters S. cerevisiae plasmids for multiple marker selection... -
CRISPR 101: Cas9 vs. The Other Cas(s)
TypeBlog Post...staggered cutter Originally classified as Cpf1, Cas12’s primary difference from Cas9 is that it generates ...j.molcel.2019.03.014 Zetsche, Bernd, HGootenberg, S., Jonathan, Abudayyeh, O., Omar, et. al. Cpf1 is a....2015.09.038 Harrington, B., Lucas, Burstein, David, Chen, S., Janice, et. al. Programmed DNA destruction by miniature... -
CRISPR Plasmids - C. elegans
TypeCollection...Cloning Enzyme(s) Delivery Resistance Co-expressed Cas9 Depositing lab Cas9 species = S. pyogenes (PAM... -
Adeno Associated Virus (AAV) for Cell and Gene Therapy
TypeBlog Post...thanks to our guest blogger, Harshana S De Silva Feelixge. Harshana S De Silva Feelixge is a researcher whose...This post was contributed by guest blogger Harshana S De Silva Feelixge. Gene therapy technologies hold...science communication. References 1. Gaspar, H. B., S. Howe, and A. J. Thrasher. "Gene therapy progress ... -
Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation
TypeBlog Post...Lin R, Wang R, Yuan J, Feng Q, Zhou Y, Zeng S, Ren M, Jiang S, Ni H, Zhou C, Gong H, Luo M (2018) Cell-type-specific...allow for dense (D), intermediate (I), or sparse (S) labeling of the cell population. Figure 2: ... -
Year of the Camelid: Antibody Style
TypeBlog Post...10.3389/fimmu.2017.01589 Jin B, Odongo S, Radwanska M, Magez S (2023) Nanobodies: A Review of Generation...https://doi.org/10.3390/ijms24065994 Xu J, Xu K, Jung S, et al (2021) Nanobodies from camelid mice and llamas... -
Plasmids 101: Biotinylation
TypeBlog Post...180. PubMed PMID: 11815306. 4. Nicole Kresge, R. D. S. and R. L. H. “The Discovery of Avidin by Esmond E... (2004). PubMed PMID: 20353954. 5. Kay, B., Thai, S., & Volgina, V. “High-Throughput Biotinylation of ...PubMed PMID: 2388593 7. Holden, M. A., & Cremer, P. S. “Light activated patterning of dye-labeled molecules...Central PMCID: PMC4461904. 9. Huang, Q., Mao, Z., Li, S., Hu, J., & Zhu, Y. “A non-radioactive method for ...PMC4439073. 12. Ludwig, L. B., Hughes, B. J., & Schwartz, S. A. “Biotinylated probes in the electrophoretic mobility... -
Viral Vectors 101: Integrase-Deficient Lentiviral Vectors
TypeBlog Post...Pike-Overzet, K., Chatters, S. J., de Ridder, D., Gilmour, K. C., Adams, S., Thornhill, S. I., Parsley, K. L.,...10.1038/gt.2009.135. PubMed PMID: 19847206 Desfarges, S., & Ciuffi, A. (2012). Viral Integration and Consequences...: 23792628 PubMed Central PMCID: PMC3773023 Howe, S. J., Mansour, M. R., Schwarzwaelder, K., Bartholomae... -
Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons
TypeBlog Post...Mahou P, Le Franc Y, Supatto W, Matho KS, Ieng S, Fouquet S, Dupin E, Benosman R, Chédotal A, Beaurepaire...technique to subdivide neural expression patterns. Hampel S, Chung P, McKellar CE, Hall D, Looger LL, Simpson ...Drosophila melanogaster. Hadjieconomou D, Rotkopf S, Alexandre C, Bell DM, Dickson BJ, Salecker I. Nature...Freundlich T, Weissman TA, Schoppik D, Wang XC, Zimmerman S, Ciruna B, Sanes JR, Luchtman JW, Schier AF. Development... -
Mammalian RNAi Tools
TypeCollection... Marmari, V., Ebrahimi, M., Rashno, M. M., Abadi, S. J., & Gharagouzlo, E. (2017). Molecular mechanisms... opens in a new window) . Rao, D. D., Vorhies, J. S., Senzer, N., & Nemunaitis, J. (2009). siRNA vs. shRNA... -
Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells
TypeBlog Post...10.1016/j.jconrel.2019.02.009 Wang D, Mou H, Li S, Li Y, Hough S, Tran K, Li J, Yin H, Anderson DG, Sontheimer...the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6 promoter-driven...Elsevier, pp 39–67 Ding Q, Strong A, Patel KM, Ng S-L, Gosis BS, Regan SN, Cowan CA, Rader DJ, Musunuru... -
No Llamas Required - Synthetic Nanobodies Against Membrane Proteins
TypeBlog Post...Stohler P, Bocquet N, Hug MN, Huber S, Siegrist M, Hetemann L, Gera J, Gmür S, Spies P, Gygax D, Geertsma ER...Breedam W, Roose K, van Schie L, Hoffmann M, Pöhlmann S, Graham BS, Callewaert N, Schepens B, Saelens X, McLellan...Egloff P, Hutter CAJ, Kuhn BT, Bräuer P, Newstead S, Dawson RJP, Geertsma ER, Seeger MA (2020) Generation... -
Chromoproteins: Colorful Proteins For Molecular Biology Experiments
TypeBlog Post....00990-19 Liljeruhm, J., Funk, S. K., Tietscher, S., Edlund, A. D., Jamal, S., Wistrand-Yuen, P., … Forster...https://doi.org/10.1007/bf00428249 Chan MCY, Karasawa S, Mizuno H, Bosanac I, Ho D, Privé GG, Miyawaki A, ... -
Truncated gRNAs for Regulating Gene Expression
TypeBlog Post...When complexed with a full-length gRNA, native Cas9's two catalytic domains, HNH and RuvC, cleave DNA strands... to dramatically alter levels of expression. Cas9’s ability to alternately serve both as a means of modifying...gRNAs An alternative approach is to modulate Cas9’s nuclease activity by modifying the gRNA with which...nucleotides of complementarity to a target site, Cas9’s DNA binding capability remains intact while its nucleolytic... -
Adenoviral Vector Production and Troubleshooting
TypeBlog Post...produce the primary recombinant adenoviral stock (rAdV-S). HEK293 cells are transfected with the AdV plasmid...The resultant lysate is your primary low titer rAdV-S, and can be stored at -80C for later use or used immediately...very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 cells and produce more...8892868. PubMed Central PMCID: PMC190817. 5. Lee, Cody S., et al. "Adenovirus-mediated gene delivery: Potential... -
Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation
TypeBlog Post...they think they are. References Berns KI, Adler S (1972) Separation of two types of adeno-associated...Maynard LH, Smith O, Tilmans NP, Tham E, Hosseinzadeh S, Tan W, Leenay R, May AP, Paulk NK (2019) Fast-Seq... . https://doi.org/10.1089/hgtb.2019.110 Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer...