...the ratio of genome-containing viral particles relative to the total number of viral capsids (which can...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...measured by qPCR.   Physical titer is typically calculated by two popular PCR-based methods: Quantitative...is a factor in AAV titration since the starting template is the purified viral vector solution (which contains...be mitigated by using purified viral DNA as the template instead of the intact AAV particle. Finally, ...absolute titer values may not be as important as the relative titer values. Finally, because physical titer ...

...L-arabinose) without signs of toxicity or burden relative to a fluorescent protein control. We also added.../L yeast extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for ... arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles in cells...from left to right: colony whiteness on inducer plates, white inclusions in cells with GVs via phase contrast...heterogeneity required single cell screening to isolate a clonal cell line capable of producing ultrasound...homogeneity, as the cells could be batch-sorted to isolate a polyclonal cell line with strong gas vesicle ...

...cells with exogenous biotin. After a few hours biotinylated proteins can be captured on streptavidin affinity... on neighboring proteins resulting in their biotinylation. The advantage of APEX is its speed, labeling...biotin catalytic domain to allow for promiscuous biotinylation. Compared to BioID, BioID2 exhibits enhanced...ways to detect RNA-protein interactions that can regulate multiple biological functions. RapID (RNA-protein...Any proteins that bind the RNA motif will be biotinylated.  Find more details about RapID! EXCELL: Proximity... Plasmid 101 Protein Expression Plasmid 101 Biotinylation Other resources Proximity labeling review:...

...undergo post-translational modification with fatty acyl chains. For instance, myristoylation requires an...Also, in cases where the protein of interest is relatively small, adding GFP may alter diffusion rates. ...Although bioluminescent probes are usually dim relative to fluorescent probes, recent engineering efforts...an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence (-CaaX... that the N- and C-terminus of GFP itself are relatively close, thereby minimizing the disruption of the...

...year. Feel the love with MTRIA sensors One of the latest additions to Addgene’s catalog is a new class of...domain of ZAP70 (ZtSH2) which binds only the phosphorylated state of an immunoreceptor tyrosinase-based...only when the RTK is activated and the ITAM phosphorylated. To demonstrate that pYtags can be generalized...smell the flowers - or rather, the lactate! The latest biosensor from Gary Yellen’s lab is LiLac, a fluorescence...concentrations of lactate. The microfluidic screening platform developed here is also a promising approach for...other targets. Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for...

...task would take longer than I thought, I’d be too late to meet up, and I’d just end up spending more time... friends, but didn’t realize it until it was too late. I didn’t spend enough time thinking about the possibilities...while the longer tasks will give you larger payoffs later. Don’t be afraid to accomplish some quick but moderately...assess whether that time was sufficient for the task later. If not, then you should definitely allot more time...friends and come back to the lab to split my cells later. Eating with friends has a somewhat immediate deadline...may have prevented me from splitting cells until late in the day anway. There are a few things I’ve found...

...caused this glowing phenomenon wasn’t until much later when, in the 1960s, Osamu Shimomura studied the ...Washington state. Using these samples, he was able to isolate two proteins from the jellyfish’s photoorgans; ... absorbs that blue light and glows green. In the late 1980s, another researcher, Douglas Prasher, got ...expressed GFP in exogenous organisms (E. coli, and later C. elegans) for the first time. After Chalfie’s ...fluorescent proteins like GFP really are. In this compilation we’ll take a closer look at how they’re used,...

...editing system is regulated not only temporally with doxycycline, but it can also be regulated spatially with...these base editors Spatially and temporally regulated AAV-based genome editing In 2016, Jonathan Ploski... mediated genome editing system that could be regulated by doxycycline. Now, they’ve developed another... that CRISPR efficiency can be controlled by manipulating complementarity between the gRNA and target....

...react and learn. All of this requires genes to be regulated dynamically.” To understand how that works in ...other brain functions, Konermann needed tools to manipulate the expression of specific genes in the brain...and genetically target them to specific cell populations. They demonstrated use of the LITEs to control...transcription in as little as 30 minutes after light stimulation. Under those optogenetic controls, gene expression...chromophores flavin-adenine dinucleotide and methyltetrahydrofolate, which are universally abundant. Second...

...thought that it was necessary to develop a single platform to connect talented professionals (who migrated...and their successes. Although all mentorship relationships are valuable and highly recommended, a mentor...mentor creates a closer and more approachable relationship for the mentee, where (s)he feels truly understood..., mentors and mentees often developed close relationships with a level of sympathy that is difficult to...confidence. These improvements, combined with the relationships fostered through the IMP have motivated many...-Harvard Medical School, where she studies age-related macular degeneration. She is also a blogger who...

...generate a knockout cell line, with the goal of isolating single cell clones to use for multiple downstream...associated with an elevated mutation burden. The CRISPR platform must be optimized to reduce off-targets and patient...Hopefully you can understand that depending on your platform, off-target effects should impact your experimental...major con is that binding by Cas does not always correlate with catalytic activity at those sites, especially...editing events will be on target. Thus, if you isolate/sequence/whatever your editing event outcome is...line to perform downstream characterization of, isolating 2 or 3 distinct clones to perform key experiments...

... colonies to isolate the monomer Sometimes a plasmid can exist in a bacterial population as a mixture ...growing the transformed bacterial culture into the late stationary phase. Growing the culture at a lower...detection methods described above can help ensure the isolation of the monomeric version of the plasmid. Linearize...ligate the plasmid If you are having difficulty isolating a plasmid monomer from a plasmid sample, or if... CP, Carnes AE. Generic plasmid DNA production platform incorporating low metabolic burden seed-stock ...

...long been extremely useful in the lab – they are relatively small and easy to work with, making them the ...much genetic and physiological data have been accumulated over the years using them. Indeed, the future...work is bright as it is now easier than ever to manipulate the mouse genome using CRISPR/Cas9. Similar ...would be great if we could just go in and quickly manipulate individual mouse genes and study their function...successful in my first attempt at using CRISPR? Later on, I found out that my experience was shared by...s41467-017-02706-7 Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-Step...

...interests are very clear, I had no idea how to translate them into real world careers. I figured this internship...but this never happened. I actually started at a later date and worked entirely virtually. Stepping into...world, I was worried about how my ideas would be translated through Zoom calls and Google docs. I pondered...Canva, and I was able to create cute and on-brand templates for these posts. Some of recent Meet an ... departments of the company. I asked questions relating to their roles at Addgene. I had a great time ...

...plastic you’ve saved. Repurposing regulated medical waste Regulated medical waste (RMV), which we collect...environmental impact of those processes? Who even regulates them? You may be surprised to learn that it hasn...Environmental Protection Agency, which started making regulations such as preventing manufacturers from dumping...and reused materials such as glass pipettes or inoculation loops. While single-use is now a popular theme...

...application we were looking at, magnetic isolation of circulating tumor cells, we found that the binding...Oxford is dedicated to generating new tools to manipulate biology based on molecular features found in ...the SpyTag/SpyCatcher system, based on a protein isolated from Streptococcus pyogenes that locks itself ...the context of cancer? Howarth: The capture of circulating tumor cells (CTCs) is going to have a big impact...

...on antibiotic plates.  Importantly, because the bacteria from which plasmids are isolated grow quickly ...easily make large amounts of plasmid to manipulate and use in later work.     How do scientists use plasmids...plasmids? Generally, scientists use plasmids to manipulate gene expression in target cells. Characteristics...

...antibiotic resistance marker. After transformation, plate the cells on medium containing the corresponding...contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing agar. Only cells... are auxotrophic for specific amino acids. When plated on media lacking the essential amino acid, the ... a toxic metabolite in gram-negative bacteria. Plating on sucrose medium will select for cells that contain...gene is used for negative selection on 5% sucrose plates. Exchange of the sacB gene for the gene of interest...

...specific, programmable platform well-suited for various innovative biological and translative technologies. Addgene...only deactivate the Type I-F system and are not translatable to the other CRISPR subtypes. Still, the large...Mali et al. in January 2013 (7, 8). Nine months later, more than 1500 publications describe work to improve...enabling access to multiple plasmids used for various platform applications. For more information on CRISPR/Cas...

...with regard to compliance with health authority regulations,” Baradaran says.  Baradaran’s path to science...is an Addgene depositor. KSQ uses a CRISPR-Cas9 platform to systematically test the function of all 20,... a genetic screen to identify the factors that regulate cell division. And that really got me hooked on...lentiviral microRNA-based system for polymerase II-regulated RNA interference. In the first two months since...also interested to see Addgene expand the sharing platform to other reagents in biology. Luk H. Vandenberghe...