...protein is cleaved by proteases. Nowadays, with ample structural information available, it’s clear that the...protein is mislocalized. The APT1-mVenus fusion is clearly a better choice to work with. Inserting a fluorescent...the existing pool of protein within a cell and may lead to over-expression artifacts. This is an important...

...Optogenetics Guide to learn more about how these modules work, and read on to learn how they are put to ...factors (GEFs) activate their partner Rho GTPase, leading to behaviors like lamellipodia or filopodia formation...these physical processes. Joachim Goedhart and colleagues in the Gadella Lab devised a set of optogenetic...

... the nonhomologous end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions...allow blocking the PAM sequence (e.g. if it would lead to a missense or nonsense mutation), blocking the...the PAM as possible (see below to find out why). Please check our recent studies for details (Paquet, Kwart...28102837. Additional Resources on the Addgene Blog Learn about High Fidelity Cas9 Variants How to Design ...

...reproducibility. These alarming results are due, at least in part, to insufficient and non-standard record...exchange information regarding lab work with your colleagues, supervisors, clients or even students, your ...track and find data and information. If an employee leaves the institution, the transition is smooth as all...scinoteELN Additional Resources on the Addgene Blog Learn about more tools for reproducibility 5 great apps...

...Addgene blog Learn about research applications of antibodies in Introduction to Antibodies Learn more about...visualization methods. Stay tuned for a future blog post to learn more about these techniques!  Direct vs. indirect...what sort of protocol you will need to use. (To learn more about antibody validation, this commentary ...

...expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific...came epigenome-engineering tools - zinc finger nucleases and TALENs fused to epigenetic modifiers enabled...Christopher Newgard’s Lab has fused LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited...comments! Find Plasmids for Epigenetic Modification   Leah Schwiesow contributed to updating this article. ...

...strain-specific tropism.” For example, AAV-PHP.B doesn’t lead to enhanced transgene expression, compared to AAV9...mice use LY6A to cross the blood-brain barrier, leading to strain-specific tropism (See S3 table (Huang... can visit our Caltech Systemic Capsids page to learn more about that collection. Table 1: Systemic capsid...systemic AAV experiments and Addgene’s AAV Data Hub to learn more about the capsid you’re interested in. Then...Fish, K. N., & Pfenning, A. R. (2022). Machine learning sequence prioritization for cell type-specific...Troiano, E. C., Moghadaszadeh, B., Peacker, B. L., Leacock, K. A., Horwitz, N., Beggs, A. H., Wagers, A. J...

...like zinc finger nucleases (ZFNs) or transcription-activator-like effector nucleases (TALENs) required...binding that positions the nuclease domains, called RuvC and HNH, to cleave opposite strands of the target... two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain... of their ability to cleave target DNA. Specifically, both RuvC and HNH nuclease domains can be rendered...SpCas9), resulting in a nuclease dead Cas9 (dCas9) molecule that cannot cleave target DNA. The dCas9 molecule...enzymes can also cleave RNAs non-specifically after the initial crRNA-guided cleavage. This promiscuous...commonly used CRISPR endonuclease for genome engineering, it may not be the best endonuclease for every application...

... and the two HindIII digested ends will anneal leaving the 5’ end of your gene just downstream of the ...require you to visualize your DNA using UV light – please note that UV light can damage DNA and that proper... colonies on the backbone + insert plate (or at least more colonies than the backbone alone plate). Unsuccessful...Blog  Additional Resources on the Addgene Blog: Learn How to Verify Your Plasmid Try Out Golden Gate or...

...reducing agent, typically containing thiols, to cleave disulfide bonds. Load the samples, one sample per...conditions from those used by the vendor then it may lead to nonspecific binding.  Whenever possible, include...in making your own knockout line for validation, please view this tutorial from Stuart Orkin's and Daniel...protein causes a thicker band, while less protein leads to a thinner band. It is important to note, however...

...multimer present in your sample. In these instances, please note that the supercoiled dimer band can sometimes... presence of a multimer in your sample remains unclear after visualizing it on a gel, one of the other...consistent with the size of a supercoiled plasmid dimer. Please note that it is common to see other plasmid confirmations...Ausubel FM. Recombination between bacterial plasmids leading to the formation of plasmid multimers. Cell. 1976...

...that introducing a Leu28Cys mutation in eDHFR does lead to covalent binding of the TMP-tag ligand (Gallagher...a different event where CP appears briefly then leaves. Images reused from (Ulrichs et al., 2023) under... if you are comfortable with it or can find a colleague to help you, synthesizing your own ligands can... D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana, R. F., Urh, M., Simpson, D., Mendez...

...prevents both the endogenous RecBCD and SbcCD nucleases from digesting linear DNA introduced into the ...E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease.  Exo will degrade linear dsDNA starting from...with these promoters (lacI, araC, cI857) to limit leaky expression of the Red system.  Using a plasmid to...the red genes needed for recombineering, but also leads to expression of the int and xis genes which are...

...Want to learn more about genome editing with CRISPRs? Check-out Addgene's CRISPR Resources Learn about...selective cassette minimizes the sequence added, leaving a 34 bp “scar”, but currently requires additional... editing and conditionally manipulate proteins. Learn more at his webpage or follow him on Twitter.   ...

...such as fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy...structure of the protein. In some cases this can lead to changes or a complete loss of protein function...more autofluorescence and nonspecific staining leading to false positives. Numerous dyes can be used for...Check out other methods using fluorescent proteins Learn more about antibodies Resources on Addgene.org ...

...epifluorescence microscopy wasn’t able to give a clear view of cellular movement in real time. Two-photon...microscopy has uncovered host-pathogen interactions leading to the understanding of effector function in infected...Weninger. "Dendritic cell behaviour in vivo: lessons learned from intravital two-photon microscopy." Immunology... Get Tips on Choosing Your Fluorescent Protein Learn Some of the Pitfalls of Working With Fluorescent...

... the pPSU2 plasmids are available from Addgene. Learn How to Analyze a Restriction Digest References ...Baby Gel Box Find 5 Smart Phone Apps for Lab Life Learn About the Evolution of Lab Techniques Resources...Kit-Free Alkaline Lysis Plasmid Miniprep Protocol Learn How to Analyze Your Restriction Digests Make Your...

... company, Science with Tom. In this podcast, we learn more about Tom and pick his brain for advice on ...importance in understanding biological history. 4. Please Don't Kill My Hive Beautifully filmed video about...generation science standards and communicate science. Learn more about Tom on his youtube channel and at sciencewithtom.com...

...Ready-to-Use CRISPR Lentivirus Learn about Other Members of the Addgene Leadership Team ....     Additional Resources on the Addgene Blog Learn More About Alex's Work with Cas9 Activators Michael...

...journey is at an end! Their name is...(drum roll, please!)... Abi! Thank you so much to everyone who submitted...Which name best fits our new immunoglobulin friend? Please help us decide by voting for your favorite on this...Original Blugene and Aavery would like your attention, please, as they introduce their new friend…  Fig...