... mismatch off-targets were only toxic if they had 5+ off-target sites. Results from the screen also reproduced...Pubmed PMID: 23394947. Pubmed Central PMCID: 4299491. 5. Bassik, Michael C., et al. "A systematic mammalian...following unanswered questions about CRISPR screen design. Unanswered questions about genome-wide CRISPR/Cas9...Morgens et al drew some generalizable conclusions about off-target activity, demonstrate safe-targeting ...

...cells using compact expression cassettes. Sci Rep 5: . https://doi.org/10.1038/srep10777  Garneau JE, ...:97–110 . https://doi.org/10.1038/s41551-019-0501-5  Liu J-J, Orlova N, Oakes BL, Ma E, Spinner HB, Baney...Addgene Blog Learn more about Cas9 Variants and the Expanded CRISPR Toolbox Read about CRISPR enzyme Cpf1 ... and specific? The two AAV strategies described above showed successful target modification, indicating...

...suggest using the imaginary quadrant to help (Figure 5). Even though this real-world data is far messier ...different populations on a plot.      Figure 5: The gating strategy from Barlow-Anacker et al., 2017...series of one- or two-marker plots, like in Figure 3 above, until the population of interest has been successfully...  Of course, there are many more things to learn about when diving into flow cytometry data. There’s different... ways to use FSC and SSC, as briefly alluded to above. There’s many variations in how one can gate, select...

...acquisition and retrieval of contextual fear memories [5].  In a modified behavioral paradigm, the authors ...primate cortex." Frontiers in systems neuroscience 5 (2011). PubMed PMID: 21811444. PubMed Central PMCID...of the important considerations you should think about when developing optogenetics behavioral experiments...scientific research is that the field benefits from collaboration and the exchange of ideas. The rapid spread ...versatility and standardization of genetic tools in the laboratory mouse, most optogenetics is performed in this...

...APOBEC-Cas9n-UGI targets its preferred trinucleotide motifs (5’-TCA-to-TUA) found within the 43 base-pair mCherry...Genome Editing. Molecular Therapy - Nucleic Acids 5:e334 . https://doi.org/10.1038/mtna.2016.48 Katti ...for base-editing activity The reporters mentioned above all rely on GFP fluorescence and cell sorting using... Additional resources on the Addgene blog Read about other CRISPR reporters Browse our CRISPR blog posts...

...conditions described above, and a short description of how the antibody performed.  Figure 5: Results for the...

... real-time PCR. Nat Methods. 2013 Oct;10(10):1003-5. PubMed PMID: 22035192. PubMed Central PMCID: PMC3216358...PMID: 20486768. PubMed Central PMCID: PMC2957240. 5.Taylor, Sean C., Genevieve Laperriere, and Hugo Germain... Addgene.org Find All Available AAV Learn More about Virus Production Find Viral Vector Protocols ...

... cells in Lennox LB (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) or on LB-agar plates containing...you enjoy playing with our genes. Have fun!    About the authors: Marjorie Buss is a PhD student studying...

... least 50bp upstream of the sequence of interest. 5. Use a silica spin column for purification of the ...and troubleshooting your DNA sequencing results Above (top right corner of this post) is an example of...

...rather than the ZymoBrothTM, so it costs us less than 5 cents per transformation. This protocol has worked...own competent cells using the protocols provided above, then you can find the subsequent transformation...

...High Throughput Capability." PLoS ONE. 3(11): e3647 (5 November 2008).  ...protein,” he says. “There is nothing you can do about it but be gentle. If it no longer works, just trash...

... Human Genetic Disease. Cells Tissues Organs, 205(5–6), 303–313. https://doi.org/10.1159/000490898 Gibbs.... J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 Idalia... more about model organisms in the first Five Popular Model Organisms blog post Read more about plasmids...characteristics that make them ideal for studying in a laboratory environment. We’ve covered five model organisms...have been specifically developed to thrive in laboratory settings. One of its most attractive features...minutes, they are perfect for molecular cloning and metabolic engineering.  The E. coli genome has an extensive...address many areas of microbial research like metabolism. Transport, biofilms, and much more. Because ...

...consumption in comparison to 37% of the general public (5, 6). Similarly 90% of active research scientists believe...activity while much less of the general public agrees (5, 6). Many of the new administration’s actions, including... with a testable hypothesis - an educated guess about how some part of the world functions. Experiments...governments exist in reality and science is how we learn about reality. Formally, a government can be defined ... More concretely, scientists gather information about reality informing people and their governments on...people to rally for science, others are concerned about possible unintended consequences of a march. Three...Democrats more often than Republicans, the March is not about Democrats or Republicans (16). This is a Pro-Science...

...rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning...hsdS20(rB-, mB-) ara-14 galK2 lacY1 proA2 rpsL20 xyl-5 leu mtl-1 Stbl4   Electrocompetent cells for cloning...thiamine metabolism Requires exogenous thiamine for growth ara disruption of arabinose metabolism pathway...little prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid... Phage resistance gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only ...repressed more   LacZ β-galactosidase activity abolished   lacY Lactose permease inactivated, lactose...plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability, reduced recombination...

...Regenerative Medicine accelerates research. Chapter 5: Research currently under way in the Kotton Lab. One...promote scientific sharing. Listen to learn more about Darrell, his research, and his focus on open science...Medicine and in the Department of Pathology and Laboratory Medicine. He attends in the Medical Intensive...NIH-funded Principle Investigator in the Kotton Laboratory and is the founding Director of the Center for...Podcast.     Additional Resources from Addgene Learn about Retinal Gene Therapy from Connie Cepko Find Plasmids...Plasmids from the Kotton Lab Learn more about Stem Cells ...

...deleting residues. Only about 10 amino acids can be deleted from the C-terminus and about 5 from the N-terminus... 153.7 (2013): 1602-1611. PubMed PMID: 23768684.  5. Thorn, Kurt. "Genetically encoded fluorescent tags...cylinder with a length of 4.2 nm and a diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel...based on cofactor binding proteins as described above. The smallest tags are peptides that are engineered...

...J Biol Chem 279.e5 (2004). PubMed PMID: 20353954. 5. Kay, B., Thai, S., & Volgina, V. “High-Throughput...cells and transgenic mice.” PNAS 100.13 (2003):7480-5. PubMed PMID:12802011. PubMed Central PMCID: PMC164612...uses of biotinylation in molecular biology! Learn about in vivo biotinylation of bacterial fusion proteins...covalently attached to the active site of key metabolic enzymes including biotin carboxylase and decarboxylases... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the...

...positive vs. negative signal remains the same (Figure 5).   A.                                                                                                        ...                                                                                                        B.      Figure 5: (A) A gate to select the negative live population...lymphocytes in a mouse tumor sample You made it! So much about the theoretical background. Let’s dive into the ...often choose a looser gate here. I am not worried about including irrelevant events in my analysis, as these...population, acquired from the previous gating pipeline, about 53% of my cells are dendritic cells and ~13% are...ve covered in this blog post. First, you learned about the types of scattering that occur when a cell is...

...promising starting point for further engineering. Figure 5: Structure-guided design and genome mining both led...Genome Mining Strategies. ACS Sens. 2022 May 27;7(5):1336-1346. doi: 10.1021/acssensors.1c02201. LiLac...screen (Koveal et al. 2022). Lactate is a key metabolite in glycolysis, so this sensor will be a valuable...valuable tool to study cellular physiology and metabolism under various conditions. LiLac has improved response...going viral! In addition to the viral preps noted above for iGluSnFR3 and JEDI-2P, several biosensors already...innovation next year! Did we miss any? Let us know about your favorite new biosensor plasmids in the comments...

...Methods. 2013 May 5:10(6):540-7. doi: 10.1038/nmeth.2450. Epub 2013 May 5. PubMed. Zebrabow: multispectral...integration of multiple copies of the construct (about 8 in Brainbow-1.0 mice.) The combinatorial effect...with the goal of overcoming the limitations listed above. First, they screened a variety of fluorescent proteins...method has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal...