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TypeBlog PostPublishedAug. 6, 2015, 2:30 p.m....where I gained invaluable experience working on and later chairing the Advocacy Committee. I’m now furthering...more. Having met many people at that conference, I later became involved with the NPA beyond the meeting....more productive and makes it easier to build relationships. Nonetheless, the committees would not function..., I had to learn how to develop new working relationships with these individuals who often had different...
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CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments
TypeBlog PostPublishedFeb. 7, 2019, 2:16 p.m....each gene, we needed to design guide RNAs, donor templates and sequencing primers for quality control. At... taken up to a month to design a single 96-well plate for HDR that satisfy all requirements. To alleviate...guides by CFD score, and it mutates HDR donor templates to avoid re-cutting when needed. It gives you ...the standard information that goes into a 96-well plate and comes out of it. Biotech companies are known... -
Starter guide to induced pluripotent stem cells (iPSCs) part 1: A renaissance in regenerative medicine
TypeBlog PostPublishedOct. 30, 2018, 1:04 p.m....of a 5-6 day old human blastocyst [6]. They are isolated mostly from abnormal and/or supernumerary preimplantation...Characteristic ESC ASC Self-renewal High Low once isolated from the human body Usage Universal. Can generate...concern High. Embryos are destroyed during ESC isolation. Low Chances of post-transplantation rejection...when used therapeutically High Low (if ASCs are isolated from the patient) For many years, ESCs or ASCs... -
The AAVantages of AAV in CRISPR Screens
TypeBlog PostPublishedFeb. 4, 2025, 2:15 p.m.... need to deliver the Cas protein altogether. Platforming AAV Interest in interrogating and mapping the...glioblastoma, and generate programmable screening platforms for mouse tissues. Your AAVancement could be next...highly scalable cell type-specificin vivoscreening platform. bioRxiv (Cold Spring Harbor Laboratory). https..., Kalamakis, G., Lischetti, U., Jabaudon, D., & Platt, R. J. (2023). Transcriptional linkage analysis ... -
Viral Vectors 101: Voltage Indicators
TypeBlog PostPublishedJan. 30, 2024, 2:15 p.m....heartbeat – a muscle contraction – is directly related to the electrical impulse of the cardiac cells....propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...serotype specificity during delivery (more on this later). Chemigenetic sensors take a hybrid approach and...JF). The voltage-dependent changes in rhodopsin modulate the degree of quenching of the fluor via FRET.... -
10 Basic tips for mammalian cell culture
TypeBlog PostPublishedJuly 12, 2018, 1:09 p.m....Thursday, or, at the latest on Friday. It’s best to split cells before the plates are more than 80-85%...Benjamin Franklin)! It’s good practice to label your plates and/or flasks with your name, date, the cell line...disorganization, apoptosis, and gene expression disregulation on HeLa cells." Journal of applied phycology27.6... -
PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs
TypeBlog PostPublishedJune 25, 2024, 1:15 p.m....2024). Chromatin context-dependent regulation and epigenetic manipulation of prime editing. Cell. https:/...primer binding site and a reverse transcription template (RTT). The design of the pegRNA has been shown...editing for precise and highly versatile genome manipulation. Nature Reviews Genetics, 24(3), Article 3. ... -
3D Printing Meets CRISPR Cas9
TypeBlog PostPublishedOct. 17, 2017, 12:55 p.m....communicate the nuances of protein structure in a flat 2-D images. In the early days of structural biology...full-color images of proteins. But they are still flat. To truly appreciate the 3D structure of these proteins... He then said “I hadn’t realized how perfectly flat the complex is on this surface”….where the complex...cleavage”. Jacob computationally added the non-template strand to the Jinek structure so that the resulting... -
Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles
TypeBlog PostPublishedJune 22, 2017, 2:30 p.m....protein or its role in a disease, researchers often isolate proteins of interest and examine them using biochemical...protein, it's important to validate its function relative to the untagged, wild-type protein. You may, for...the protein of interest and then analyzing the relative colocalization of their fluorescent signals and...One should also be aware of additional problems related to the properties of FPs themselves. These can ... -
Google Forums Round Up: First Impressions of NgAgo
TypeBlog PostPublishedAug. 4, 2016, 2:30 p.m....institutions recently reported their inability to recapitulate the results of Gao et al 2016 in a letter to...NgAgo DNA guide is inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can... the instability of the supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly degraded... an ssDNA guide. It is possible that, during translation, only a small fraction of nascent NgAgo protein... -
PITChing MMEJ as an Alternative Route for Gene Editing
TypeBlog PostPublishedFeb. 23, 2016, 3:30 p.m....double-stranded break. HDR copies the sequence from a repair template with flanking sequence homology for error-free...systems, and NHEJ will be favored even if a repair template is present. HDR also presents a cloning dilemma...CRISPR gene editing. In organisms where HDR is downregulated, MMEJ represents another method for making targeted...’s become clear that the power of this editing platform lies in the diversity of nucleases and their applications... -
Tips for Using BLAST to Verify Plasmids
TypeBlog PostPublishedMay 29, 2014, 1:29 p.m....blastn), Standard Protein BLAST (blastp), and Translated BLAST (blastx). NCBI has a terrific getting-started...gaps in the nucleotide sequence, you can run a Translated BLAST (blastx) search to check for expected ORFs..., due to the additional searches involved in translating the nucleotide sequence into all six possible...choose for your sequencing result and can easily translate it, we recommend using blastp over blastx. The... -
CRISPR 101: RNA Editing with Cas13
TypeBlog PostPublishedJuly 31, 2020, 12:30 p.m....would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...would permit point mutations in RNA which could recapitulate or rescue known pathogenic alleles, or introduce...ADAR will preferentially edit an adenine if the template has a cytosine mismatch at that position. PspCas13b-ADAR2DD...RNA Editing Capabilities through RESCUE Two years later, the Zhang lab expanded the RNA editing toolkit ... -
AAVs in Retinal Gene Therapy
TypeBlog PostPublishedJuly 31, 2018, 12:55 p.m....field to a grinding stop and only now, 2 decades later, is it finally back in the limelight. On December...and severe visual impairment that faithfully recapitulated the human condition. Luck brought all these ...X-linked retinitis pigmentosa, achromatopsia, and age-related macular degeneration are ongoing and some will ...capsid variants, designing expression cassettes to regulate and optimize transgene levels, and large scale... -
The Importance of a Fun Workplace: Company Culture at Addgene
TypeBlog PostPublishedSept. 3, 2015, 2:30 p.m....trivia, and everything in between). Eleven years later, Addgene is a company of fifty instead of three...situation- do we work so hard so we have time to play later? Or, does our play encourage us to work hard? Whatever...employees. The teambuilding culture helps us build relationships and thereby work better together.I realize I...encourage everyone to make an effort to build relationships with their colleagues. It can be easy to get... -
Using AAV for Neuronal Tracing
TypeBlog PostPublishedAug. 9, 2018, 1:04 p.m....bi-directionality. For predominately anterograde tracing, biotinylated dextran amines (Veenman et al., 1992) or fluorescent...., 2007; plasmids at Addgene). AAV vectors: A platform for delivering neuronal tracers AAV vectors have...overview see Addgene’s AAV guide. AAV particles are relatively small (20 nm) in size which makes them capable...Transport: Cargo-Specific Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356...Veenman, C.L., Reiner, A., and Honig, M.G. (1992). Biotinylated dextran as an anterograde tracer for single-... -
Supporting Reproducibility with a Connected ELN
TypeBlog PostPublishedJan. 17, 2019, 1:38 p.m.... added on a regular basis, so the ecosystem of related tools is always growing. Beyond the ability to ...formatting. In this way data is moved from a static and isolated vehicle and becomes part of the dynamic and connected...understand and/or attempt to reproduce research relating to data deposited into a repository access to ...can be viewed – and conceivably additional work relating to reproducing the original research can be documented... -
An Inside Look into Addgene's Dev Team
TypeBlog PostPublishedJan. 23, 2020, 2:45 p.m....to be translated into Modern Greek to be easily accessible today. Roughly speaking, we translated our software...sprints is themed and the stories in that sprint relate to the theme. For example, we could have a shipping...feature to work. We will then go back to this area later and tidy it up. Conclusion While none of us are... -
The PAM Requirement and Expanding CRISPR Beyond SpCas9
TypeBlog PostPublishedAug. 20, 2020, 2:30 p.m....binding specificity: SpCas9 variant Mutations (relative to SpCas9) PAM sequence D1135E variant ...bacterial species Many more Cas9 orthologs have been isolated, and our understanding of their PAMs is shown ...same species. Expanding the CRISPR toolbox The isolation of novel CRISPR proteins has and will continue... an intrinsically high-fidelity genome-editing platform. Genome Biol 19: . https://doi.org/10.1186/s13059... -
Twenty Years of Sharing: Expanding Accessibility
TypeBlog PostPublishedNov. 21, 2024, 10:47 p.m....available to many countries, import and export regulations, differing customs processes, and the distances...through the use of popular Chinese social media platforms. As you can imagine, shipping to so many countries... a Primer Creating Bacterial Glycerol Stocks Inoculating a Liquid Bacteria Culture Collections ... Primers for PCR Agarose Gel Electrophoresis Inoculating Liquid Bacterial Cultures Restriction Digest ...