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...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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...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 ...

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...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...

...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...

...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...