...Identifying which genes are important for a phenotype can lead to a wealth of additional experiments investigating...consists of two components: the non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA...to enrichment or depletion of specific gRNAs can lead to skewed results. Selecting a cell type - Theoretically...validation practices, CRISPR libraries can help you learn a lot about your phenotype of interest. For more...Lentiviral CRISPR Libraries” (McDade et al., 2016). To learn a bit about how CRISPR/Cas9 can be used in other...

...dependencies  Take, for instance, a recent preprint released by the Boehm and Beroukhim labs at the Broad Institute...genetic dependencies of individual tumors, which can lead to new cancer targets and related biomarkers . Data...strongly correlated genetic dependencies that were clearly associated with European or East Asian ancestry... accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’...undercounted by approximately 20%. In some cases, this may lead researchers to deprioritize genes that could be ...

...our CRISPR Featured Topic Page Learn about RNA Targeting with Cas13a Learn about CRISPR DNA Base Editing...contain the RuvC and HNH domains responsible for DNA cleavage, so they cannot directly edit the genome. A Cas13...They hoped to find a stably folded ortholog that cleaved RNA robustly, unlike LwaCas13a, which must be stabilized... multiplexed manner to edit multiple RNAs. It’s clear that CRISPR RNA editors will open new doors for ...

...cells are lysed, RNAs and proteins are removed to leave you with DNA. This DNA is then washed, and either...purify DNA, cellulose also efficiently binds, or at least entraps nucleic acids. The Botella lab took advantage...around. Finally, purified total nucleic acids are released by dipping the cellulose directly into a PCR buffer...columns, suggesting that columns can be reused at least five times. DNA purification kits don’t come with...used plasmid purification columns could also be cleaned with 1M phosphoric acid and re-used, but trace ...

...and mascot Blugene (check back on December 5 to learn more about this!). I’ve assembled a series of screenshots...filter, this one from the ‘Requests’ category, will leave us with a single plasmid to consider. If you get...search. Simply click the ‘X’ next to any filter to clear it. Here’s a list of available filters to refine...great improvement for our website and we hope it leads you to exactly what you need with every use. If ...redesign. Additional resources on the Addgene blog Learn more about other features on Addgene's website ...

...your cells of interest. Cas9 RNPs are capable of cleaving genomic targets with similar efficiency as compared...levels shortly after transfection, and are quickly cleared from the cell via protein degradation pathways....known as nucleofection, which forms pores in the nuclear membrane, allowing for entry of a DNA template..... "Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing." Journal...the Addgene Blog Subscribe to CRISPR blog posts Learn about other CRISPR delivery techniques Find tips...

...contributed by guest blogger, Luke Lavis, a Group Leader at the Janelia Research Campus, Howard Hughes Medical... photoactivation, etc. These impressive advances lead to an obvious question: In this age of GFP and its...fluorophore can emit a limited number of photons before bleaching. The amount of information one can extract from...Luke D. Lavis. Luke D. Lavis is currently a Group Leader at the Janelia Research Campus, Howard Hughes Medical...PMC3246539. Additional Resources on the Addgene Blog Learn About Other Protein Tags Catch Up on Your Fluorescent...

...Resources on the Addgene Blog Learn to do Restriction Cloning Try SLIC cloning Learn about Gibson Assembly ... One key difference is the plasmid DNA must be released from the bacteria in order to serve as PCR template...reaction. You just need to lyse the bacteria to release the plasmid DNA by either briefly boiling the sample...Read about Choosing a Molecular Cloning Technique Learn to Perform a Bacterial Transformation Deposit Your...

...visualizing promoter activity, and much more. Read on to learn more about GFP, how scientists have evolved this...exist with only slight variations separating them.  Please note that many fluorescent proteins found on the...F64L; S65T, Y66W; N146I; M153T; V163A; A206K Cerulean F64L, S65T, Y66W, S72A, Y145A, H148D, N149I, M153T... Y145A and H148D Stabilizes the structure of Cerulean derivatives F99S, M153T, V163A Improves folding...visualize the effects of alterations to this activity. Learn more about optogenetics at OpenOptogenetics and ...

...CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi...separated multiple sgRNAs with tRNAs, which are cleaved out of the RNA transcript by endogenous processing...and found the protein La (a small RNA-binding exonuclease protection factor) promotes editing (Yan, J et...hypothesized that the improved efficiency is at least in part due to La binding and stabilizing the 3’...Neurodegeneration Collection updates Last but not least, check out the new and noteworthy tools featured...

...each viral lot. To learn which specific QC experiments were performed on your lot, please contact us. Full...medium (OptiPro SFM) containing lentivirus is first cleared by low-speed centrifugation and then by filtration...responsive element (RRE) gene and the cellular ribonuclease P/MRP 30 kDa subunit (RPP30) as a control for...the sample from the DNA tube all the way to the cleared viral preparation pool. In addition, the final ...

...replicon of pTargetF may exhibit a more serious leaky expression in BL21(DE3), which we presume caused...that indeed the gRNA-pMB1 had a slightly higher leaky expression in BL21(DE3) that resulted in a lower...a sacB gene renders it beneficial for plasmid clearance. It is more convenient to update the spacer(20nt...Anhydrotetracycline (aTc) induces expression transposases leading to increased integration frequency. (C) Increasing...rounds of transfer on transposition-inducing agar lead to higher cargo copy numbers. (D) The effect of ...

...integrating neuronal signalling and triggering release of the stress hormone norepinephrine to promote...are chemogenetically activated, norepinephrine release causes a freezing response, a form of immobility... the NAc, where enhanced glutamate transmission leads to locomotion sensitization. (for review see van... as sleep, food intake, mobility/coordination, learning, and memory. We’ll cover these in a  future blog...Guide to Viral Vectors Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...tips from an AAV rookie  Learn about some considerations when using AAVs Learn more about using AAV as ...complex (LY6A) as a receptor for PHP.B. Read on to learn how the design of PHP.B may have contributed to ...182-188. PubMed PMID: 29175632. If you want to learn more about how PHP.B and PHP.eB were engineered,...PMC5088052. Additional resources on the Addgene blog Learn more about PHP.eB, and PHP.S, a peripheral nervous...

...Blog Learn All about Mammalian Vectors Read up on CRISPR Delivery Systems for Mammalian Cells Learn about...older than those available from biorepositories leading to genotypic and phenotypic artefacts. The life...commercial repositories and the same cell line from a colleague?”. The main difference is the QC and validation...usually the case if you obtain a cell line from a colleague. Time and money are wasted, papers are retracted...

...remains unclear whether the FP misfolding impacts fusion protein function and this could lead to under...localize [4]. Most investigators are surprised to learn that FPs are often poorly suited for cellular compartments...glycosylation, disulfide bond formation, and proteolytic cleavage. FPs localized to organelles are equally susceptible... dark fluorescent proteins in your experiments, please consider the following: How can one ensure that...

...bit of a steep learning curve and many opportunities for mistakes. The easiest way to learn optogenetics... is an approximation of reward or the sense of pleasure that the animal perceives through taking a drug...University.  His interests include the role of learning and memory in opioid addiction and science communication...Kheirbek, Mazen A., et al. "Differential control of learning and anxiety along the dorsoventral axis of the...

...different chromatin states. Alexander Stark and colleagues at the Research Institute of Molecular Pathology...Initiative, led by Dominic Esposito, has recently released a collection of 360 Ras pathway plasmids to the...Drosophila cells, targets 13,501 fly genes with at least three independent gRNAs, each chosen strategically...vacuolar) pH environments that inactivate SEP and leave only DsRed to fluoresce (1). With variants that ...effects on gene regulation. Professor Zoldos and colleagues from the Department of the Biology, Division ...26912792 FusX-TALEN assembly system TALEs as nucleases (TALENs) are genome-editing tools that are deployed...auxotrophies or using different auxotrophic backgrounds can lead to disparate physiological effects and complicate...

...Bench series was born from the shift to remote learning due to COVID-19. We had many protocols for plasmid...details about personal protective equipment to learning about how to use a tissue culture hood and other...other lab equipment, we’re here for you as you learn the ropes! All material can be found on our Protocols...Protocols page and YouTube page.  Happy learning, and welcome to the lab bench, new scientists!   ...

...warm meal on a winter day, you should be happy to learn that many DNA-based reagents are named for soup ... and non toxic to cells. Because it rapidly photobleaches, DFHBI can only be used for qualitative, but...regions in the 5’ UTR of mRNA that bind metabolites, leading to conformational changes that control the expression...the Corn-DFHO complex. For dessert (last but not least), the only fruit-named aptamer, Mango, binds to ...PMC4143336. Additional resources on the Addgene blog Learn more about aptamer fluorophores Investigate other...