...at East Tennessee State University. He works with gene expression in Pseudomonas aeruginosa. Follow him...Protocols Check Out How Addgene Develops Lab Management Software Resources on Addgene.org Find Protoctols ... and do not necessarily represent the views of Addgene. I think it’s safe to assume that anyone reading...by New England BioLabs In my lab we do a lot of genetic construction and cloning experiments. Not all of...100percentSDS.     Additional Resources on the Addgene Blog Learn How to Keep a Lab Notebook for Bioinformatic...

...Plasmids for Creating and Controlling Gene Networks Browse All SynBio Plasmids ... education policy. Additional Resources on the Addgene Blog Synthetic Biology and the Importance of Assembly...All Synthetic Biology Blog Posts Resources on Addgene.org Guide to SynBio Assembly Standards Find Plasmids...

...telephone, until the message reaches the nucleus where gene expression changes lead to cell proliferation and... on Addgene.org Find nanobody expression plasmids here Learn more about optogenetics in Addgene's optogenetics...induced binding. The lab used this general design scheme to generate two more OptoNBs, one against mCherry...loop 1 always generated OptoNBs with dark-induced binding, but insertion into loop 6 generated both blue ...on the key components of OptoNBs, how they are generated, and their use for protein purification and controlling...nanobody’s ability to bind its target protein.  Generating OptoNBs The opening and closing of the AsLOV2...spatiotemporal resolution than pharmacological or genetic manipulation of the signaling pathway.  As a proof-of-principle...

...endonucleases. A given prokaryote typically has genes encoding one or a few restriction modification systems...Results Use REPLACR Mutagenesis to Mutate Your Plasmids Resources on Addgene.org Check Out Our Plasmid...newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions is the...cleave DNA. DpnI is often used for site directed mutagenesis. During this process, incorporation of a desired...mutation into your plasmid of interest by PCR generates mutated plasmids with no methylation (there are...illustrates how sequence visualization software (we used Snapgene here) can help you assess whether your restriction...describing dam-/dcm- E. coli Resources on the Addgene Blog Keep up with the Latest Cloning Techniques...

...The first base editors revolutionized CRISPR gene editing. Cytosine base editors (CBEs) and adenine base...conducted a CRISPRi screen to identify endogenous genes that impact CGBE efficiency. In addition to using...review how CRISPR-derived base editing works in general. Base editors use either catalytically dead Cas9...efficiency and produced fewer indels than first-generation editors. This team also demonstrated that different...sequences) (Huang et al., 2024). BE4max, a CBE, generated similar deletions at a lower frequency. Their ...and its inclusion significantly reduced indels generated by CGBEs. What's next? Base transversion editing...base transition editing, we can expect several generations of optimization before the field begins to converge...

...where we could incorporate cold-tolerance inducing genes within a regular mesophilic host system. This would...resources on the Addgene blog Find our synthetic biology blog posts Resources on Addgene.org Find plasmids... pC013-Twinstrep-SUMO-huLwCas13a plasmids from Addgene, which would potentially allow a stable Cas expression...” by Benoit Giquel, an outreach scientist from Addgene. The talk highlighted the importance of reagent...

...In this case, Konermann et al. targeted MAPT, the gene encoding dementia-associated tau, delivering dCasRx...possibilities. To identify Cas13d, both groups used very general bioinformatic screens that looked for a CRISPR ...scientific sharing will continue to grow, and we at Addgene appreciate our depositors for making their tools...29551272 Plasmids from this paper are available at Addgene. Yan, Winston X., et al. “Cas13d Is a Compact ...29551514 Plasmids from this paper are available at Addgene. Learn more about research at Arbor Biotechnologies...Biotechnologies. Additional Resources on the Addgene Blog: Learn about CRISPR RNA Editing with REPAIR Get tips...Cas13a (C2c2) Characterization Resources on Addgene.org: Find RNA Knockdown Plasmids Check out our CRISPR...

...situ is the knock-in of fluorescent tags into the gene locus of interest. An advantage of this alternative...studied a synaptic protein that is linked to neurodegeneration. The level of this protein is decreased in...PMCID: PMC4121734. Additional Resources on the Addgene Blog Fluorescent microscopy techniques - Which... tagging strategies Additional Resources on Addgene.org Fluorescent protein plasmids Use FRET to study...protein-protein interactions Deposit plasmids with Addgene! ...

... widely available and applied by researchers for gene targeting. Fast forward a decade, and the CRISPR...Resources on Addgene.org CRISPR software and resources Addgene CRISPR Guide Addgene’s CRISPR 101 eBook... trans activity. AsCas12a, on the other hand, generates staggered DNA nicks at its target in cis and cleaves...end goal. CasPEDIA also features useful links to Addgene resources throughout the site. In the Experimental...each enzyme page, you'll find a link to search Addgene's collection for plasmids containing that enzyme... from the article if they're available through Addgene. So whether you're looking for the exact Cas9 plasmid... or browsing for the latest AsCas12a plasmids, Addgene and CasPEDIA have got you covered!     ...

...repair of Cas9-generated breaks is useful if the intent is to make a knockout in your gene of interest, ..., as it is prone to generating indel errors. Indel errors generated in the course of repair by NHEJ are...are typically small (1-10 bp) but extremely heterogeneous. There is consequently about a two-thirds chance...Of some importance, the deletion can be less heterogeneous when constrained by sequence identities in flanking...overhangs is available. Cas9 can also be altered to generate a targeted single strand break; when two such ...  Dale Ramsden is a member of the curriculum in Genetics and Molecular Biology, the Dept. of Biochemistry... Break Diminishes Its Mutagenic Potential. PLoS Genet 5:e1000683 . https://doi.org/10.1371/journal.pgen...

...Ready-to-use AAV available from Addgene's viral service encoding biosensor tools, including calcium sensors...

...from Jiazhang Lian’s lab that enables simultaneous gene activation, repression, and editing with a single...studies.    Duong et al., Genetics 2021. https://doi.org/10.1093/genetics/iyab006       The CRISPR ... need. Here's what you'll find in this post: Epigenetic engineering with single-pot assembly Tracking...CRISPR plasmids New items from the viral service Epigenetic engineering with single-pot assembly By Eric ... of plasmids to the repository that will help epigenetic researchers custom design their own engineering...) Golden Gate assembly (backbones available at Addgene) with examples of potential synthetic chromatin...with BFP, sender cells expressing surface BFP generated BFP-labeled receiver cells. What’s more, G-baToN...

...downstream application such as quantitative PCR to assess gene expression. There are several things that researchers...everything, including your skin. Here are some general tips for working with RNA, even if you are using...10.1038/nprot.2006.83 Additional resources on the Addgene blog Read blog posts related to plasmid protocols...for purifying DNA without a kit Resources on Addgene.org Check out our molecular biology protocols Find...

...You realize, much too late, that you labeled your gene of interest incorrectly on slides 8-16. Your notes...remember related to this paper and/or biology in general until I reached Fig. 6, Panel L, and hope nobody...breath–you got this!  Additional resources on the Addgene blog Transferable Skills Guide: Time Management...

... useful as a mechanism of transient knockdown of gene expression. This system could be used in lieu of...complementary site in the genome where it will proceed to generate a blunt DNA double strand break. Cas9 has been...Cas12’s primary difference from Cas9 is that it generates staggered breaks, also known as sticky ends. Cas12a...targeting experiments requiring single strand DNA generation, such as homology directed-repair. Cas12a is ... of DNA. This makes it particularly useful in generating transient edits of signaling molecules (RNA) ...If your genome engineering goals expand beyond generating frameshift mutations or single base pair edits...s41586-021-03886-5 Additional resources on the Addgene Blog CRISPR 101: Which Cas9 do I choose for ...

...Chemical Modifications of CRISPR RNAs to improve Gene-Editing Activity and Specificity. JACS 144, 12584...resources on addgene.org  View Addgene's collection of empty gRNA vectors View Addgene's CRISPR resources...effectively serving their function of guiding Cas9 to generate a targeted break. These advances in gRNA technology...or immunity itself is being studied. Check out Addgene’s validated gRNA repository to find pre-tested gRNA...photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable 2-nitrobenzyl...lentiviral plasmids Additional resources on the Addgene Blog  How to design your CRISPR gRNA CRISPR 101...

...have been modified for genome editing, activating gene expression, visualizing genomic loci, and much more...plasmids from this publication at Addgene. Resources on the Addgene Blog Read about Cas9 Homolog Cpf1...effect on specificity was similar; SpCas9-HF1 generated fewer off-target cuts when compared to WT SpCas9...collaboration with the Joung Lab, they used targeted mutagenesis to create HypaCas9 (SpCas9 N692A/M694A/Q695A/...-HF1. The HypaCas9 plasmids are available from Addgene here! Future possibilities for Cas proteins With...26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure...26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading governs...

...cells)  that allow for continuous expression of genes integrated into the insect cell genome. Both of ...and cell free expression systems. Overall the general strategy for protein expression consists of transfecting...systems Yeast are a great expression system to generate large quantities of recombinant eukaryotic proteins... species due to its use as a model organism in genetics and biochemistry. When using S. cerevisiae, researchers...large-scale, stable production of proteins. In general yeast expression systems are easier and cheaper... use. Alyssa D. Cecchetelli is a Scientist at Addgene. She received her PhD from Northeastern University...proteins in plant root exudates Resources on the Addgene Blog Learn about inducible promoters Learn about...

...fewer transcripts of their antibiotic resistance genes and can therefore be cultured in media containing...plasmid prepping is a breeze. You get your stab from Addgene, streak for single colonies, sub-culture, and prep...use a different strain. For most applications, Addgene uses and recommends DH5𝝰! This strain consistently...density and plasmid yield as these factors are generally, but not perfectly, correlated. Typical growth...sufficient gas exchange in the overnight culture. Generally, large cultures in flasks may be shaken around... strain instructions from the manufacturer. On Addgene plasmid pages, you may see depositor recommendations...writing of this post. Additional Resources on the Addgene Blog Visit our Plasmids 101 Topic Page Learn How...

...error than normalizing to so-called housekeeping genes (Aldridge et al., 2008).  Woah! That was a lot of...quantification Normalizing to ubiquitously expressed genes Not as accurate; increased antibody costs; increased...Antibodies Resources on Addgene.org Addgene's Western Blot Protocol Addgene's Western Blot Protocol Video...Looking for a western blot protocol? Check out Addgene's western blot protocol and protocol video!  Antibodies...gel. Handcast gels are significantly cheaper and generate less waste. However, they also add time to an ...in size. However, these systems are expensive, generate plastic waste, and limit the amount of optimization... can only compare samples to a standard curve generated on the same blot. If your protein of interest ...