...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines already expressing Cas9...

...ll need a few controls specific to flow as an application. These controls will allow you to distinguish...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...commercial antibody pages. Flow cytometry can be a complicated and daunting process. Controls like single color...research technician in Arlene Sharpe's lab at Harvard Medical School.   More resources on the Addgene blog Introduction...

...of interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism... for example).  Given that the liver is an immunologically active organ, this advance is likely to afford...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...clonal multiplexing of cells in live imaging application. Each visual barcode consists of a fluorescent...

...neurons and specific behaviors. Chemogenetics uses genetically engineered receptors to interact with small molecules... to optogenetics but instead of light, these genetically modified receptors are activated by small molecules...some GPCRs further to respond to specific, pharmacologically-inert, small molecules. These new receptors...Kadowaki S, Haga T, Takaesu H, Mitaku S (2002) Identification of G protein-coupled receptor genes from the...

...α-complementation. The system described above was put to practical use in the following way. Scientists engineered...insert. All colonies on this plate should be blue, indicating that your IPTG and x-gal are working as they ...of false positives: Blue-white screening only indicates the presence of AN insert, not necessarily YOUR... Jessica Welch...

...missing or damaged nucleotides that are common to biological sources of DSBs, and which need to be processed...generated in the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There.... NHEJ can also be engaged by variants of the canonical Cas9 approach. A pair of CRISPR guides that flank...Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular Cell 55:829...

.... Listen to our podcast interview with Harvard Medical School researcher Connie Cepko to learn about her...mentoring programs. The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet...This is a chance to practice leadership and communication skills. The leader also provides materials such...present formally often enough) and give honest, critical feedback for improvement. Go to a science or career...

...produce genetically modified cells in less than a month's time. Addgene: What are the implications of this...Musunuru: The kit includes 832 plasmids containing prefabricated pieces that allow you to build a DNA-binding...diseases. We are knocking out genes that have been implicated in disease or, in cases where particular variants...

...time it takes to make a plasmid could increase dramatically if you are piecing together several PCR products...to make plasmids that already exist, and we significantly reduce costs of producing plasmids with high-throughput...The faster methods, like Gibson assembly, are typically more expensive, so you’ll have to find a balance...calculations for any of the numbers in this post is a mathematical story well beyond my ability to tell. But even...

...protein expression in mammalian cells: easy to use biological tools are abundant! In this post, we will review...that! CRISPR interference (CRISPRi) targets a catalytically dead Cas9 fused to a transcriptional repressor... protein, then these systems need to be site-specifically knocked in. This can be labor intensive and ...and target-specific protein degradation. Nature Chemical Biology. May 2018. 14(5): 431-441. PubMed PMID...

...donating and receiving labs)” — Jim Woodgett, biomedical researcher at Lunenfeld-Tanenbaum Research Institute...reagents and protocols so easy!” — Nina Jain, Harvard Medical School. We want to give a special thanks to the...decade. We did this the old-fashioned way: through physical outreach, visiting organizations, knocking on ...standardized agreements and electronic signatures to dramatically speed up MTA approvals. In 2013 we were thrilled...

...our second example we’ve added some simple but critical logic. Some steps fail “hard”, they reset the ...interchange them easily as needed. In this case, the physical considerations of the laboratory make object composition...science_name`. You'll need to decide given the technical details of the problem you're solving, but consistency...interesting biotechnology code.There are valid technical reasons to choose a non-Python stack, but if you're...

...choose well. What are their qualifications? Do they have certifications? How can they demonstrate their...help you. A good coach usually will be a clear communicator and make direct suggestions for improvement ...she is especially excellent in coaching for communication skills. Industry transition is also an area ...

...-humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 ...ecotropic or amphotropic MuLE lentivirus provides significant genetic power and allows high throughput genetic...be used to construct complex lentiviruses for modification of mammalian cells with a single viral infection...Gammaretroviral Vectors: Biology, Technology and Application. Viruses. 2011. PubMed PMID: 21994751. PubMed...

...engineered microbes, used coffee grounds could be economically repurposed as starting material for the bioproduction... FLINC biosensors for live-cell imaging of biochemical activity Article contributed by Angela Holmes... live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence ...peak of 519 nm. These proteins have improved photophysical properties and can be used in quenching, FRET...

...can contain different neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin... show a high proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in ...in high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...Hybrid for Multiplexed Editing and screening Applications (CHyMErA) Knockout Libraries express hybrid ...

...methods greatly improved CRISPRa, expanding its practicality and popularity in research (Tanenbaum et al....themselves. SunTag, SAM, and VPR have all shown significant improvements upon the initial dCas9-VP64 method...Performance Generally, VPR is found to have significantly higher activation levels than the initial dCas9... (2014) A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging....

..., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered that...viral particles that have the gene deletion could replicate when challenged with the S. islandicus CRISPR-...Hematopoietic Stem Cells. Molecular Therapy - Methods & Clinical Development 9:390–401 . https://doi.org/10.1016...B, Bondy-Denomy J (2020) Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies....

...ampicillin resistance cassette and an origin of replication, which are not schematized. Sizes of fragments...validated and were chosen because of their broad applicability in different model systems. What’s the error...five months. When they tested those clones (in duplicate), 93% had the correct banding pattern in the restriction...correct. Because they created the assemblies in duplicate, they were able to confirm that at least one plasmid...

...its design changes for every new prime editing application. And optimizing its sequence is important, as... gRNA (Anzalone et al., 2019), disrupting its canonical structure and Cas9 binding (Nishimasu et al., ...other bases in this location bind to Cas9 with canonical sgRNA structure (right). Created with BioRender.com...structured motifs like mpknot, use the pegRNA Linker Identification Tool (pegLIT) to create linkers that minimize...