...Gag-Pol polyprotein, cleaves the proteolytic site connecting the two proteins, thus releasing Cas9. Nanoblades...Additional resources on the Addgene blog Learn about RNP delivery Learn about CRISPR delivery methods for mammalian...CRISPR guide Find CRISPR plasmids and resources Learn about viral production ...

...Cell-penetrating Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess...variants By Mike Lacy In 2022, Atsushi Miyawaki and colleagues identified a highly bright and stable fluorescent...just a few days later, Mohan Balasubramanian and colleagues showed the single mutation E138D is sufficient...is worth mentioning that Kiryl Piatkevich and colleagues generated yet another monomeric StayGold variant...-3188559/v1.  Neurogenetic alchemy: CAP-Mac unleashes the secrets of primate brains with brilliance ...

...previous post from our Plasmids 101 series, we learned how the Cre-loxP recombination system can be used...DNA fragment using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment...reporter will be greatly reduced, allowing for clearer characterization of the location, morphology, and...containing FLEx technology. Read the genOway press release for more information. References: 1. Schnütgen ...Means of Genome Engineering Using FLEx AAV Vectors? Learn about Viral Vector Elements Read Our Primer on Optogenetics...

...Chromoproteins also enhance corals’ resistance to mass bleaching, the loss of their symbiotic algae, during heat...rhamnose-inducible promoter. The resolution step leads to the desired mutation and excision of the vector...mammalian cell expression here   Now that you’ve learned what chromoproteins are and how they can be used...blog Find our Fluorescent Protein 101 blog series Learn more about FRET Find protocols and tips for making...Blanquet RS, Phelan MA (1987) An unusual blue mesogleal protein from the mangrove jellyfish Cassiopea ...

...hatching into their adult phase. It is important to clear out the parent crosses from the container before...sharing-oriented, and there are many resources to help you learn more about fly biology and fly-based experimental... and gene expression. In the next post, you’ll learn the fundamentals about how many of these resources...between pathogens and their hosts. It’s not always clear which side he roots for. You can try to convince...Methods for Site Specific Recombination in Drosophila Learn about Cre-Lox Resources on Addgene.org Find Drosophila...

...experience, I was still incredibly nervous about leaving academia. A lot of academics tend to view industry...postdoc experience, I’m going to share what I’ve learned and hopefully make your decision a little easier...publications, which may take a longer time to acquire. Learning about drug discovery: Unless your academic lab...market usually takes over a billion dollars and at least a decade of work. The number of steps, decisions...an academic postdoc, you’re often required to at least attempt to write grants. If you don’t enjoy or excel...

...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...

...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 ...

...Previously developed genome editing methods using nucleases and base editors have the potential to correct... extending the pegRNA primer binding site to at least eight nucleotides enabled more efficient prime editing...precise changes. However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR...base in genomic DNA without double-stranded DNA cleavage." Nature 533.7603 (2016): 420. PubMed PMID: 27096365...on the Addgene blog Visit our CRISPR topic page Learn the basics of CRISPR Download the CRISPR 101 eBook...

...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...

...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 ...

...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...

...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...

...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...