...Check out this post for a refresher on AAV titers Making contact with a cell Although an individual AAV ... this type of vector to such a high titer when looking for effective gene expression. How do you achieve... Hanley is a Research Team Lab Tech at Addgene working on methods to improve AAV production.     References...

...hot plasmids articles. Think we missed something smokin'? You can pitch a Hot Plasmids section (antibodies...heatshock promoter) drives expression of the tumor-seeking CAR (chimeric antigen receptor), whereas a ubiquitous... variants with similar capabilities. If you’re looking for a super stable FP for your next microscopy ...

... apart sources of information they don’t like, looking for reasons to dismiss them. There’s evidence to...Zealand’s multiple Covid-19 lockdowns, I held mask-making workshops in my community. I brought along printed... to better meet the needs of the people we are talking to. Emma Harcourt is a PhD student in the Department...

...idea to take some time to confirm that you are working with the correct construct, and verify that the...Consult the manufacturer’s manual for the optimal working conditions for each enzyme. Watch out for methylation...run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA is isolated and run on ...

...— by directly typing terms in a search bar, by ticking some boxes, by selecting a suggestion — how should...resources. Filters We identified the most useful kinds of information per resource category to expose as...our search indices in order to support different kinds of matches: exact words, partial words, phrases,...

...great? It produces a ~10 fold higher titer virus, making it ideal for CRISPR pooled library screening experiments...V2.0 was made available through Addgene in 2015, making this the most recently available plasmid in the... thank all of our depositors and requestors for making their reagents available and for supporting scientific...

...question that didn’t get answered live - but he kindly took some time to address all your unanswered questions...astrocyte specificity and would like to get it working in microglia as well. A:  We are not aware of any... tail vein injections but would like to avoid "leaking" into the brain, would you recommend using the ...

..., you may be curious about all of the different kinds of antibodies that are available. One common type...References Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B,...TA, Humphries MJ (2016) Ligand-induced Epitope Masking. Journal of Biological Chemistry 291:20993–21007...

...reference protein) under identical conditions, a ranking of the practical brightness can be made (Goedhart...repeating this with another fluorescent protein, a ranking of practical brightness can be made. This strategy...their practical brightness in a system closely mimicking the future application. In figure 2 we provide...

...high-throughput manner and therefore push us closer to making cancer precision medicine a reality. Challenges...Since time is of the essence in clinical settings, looking ahead, we’re starting to build saturating libraries... Institute of Harvard and MIT and is focused on making precision functional genomics a reality for future...

...These enzymes also do not require a protospacer flanking sequence, so you can target virtually any RNA ...Cas13d enzymes have a median length of ~930aa - making them 17-26% smaller than other Cas13s and a whopping...and we at Addgene appreciate our depositors for making their tools available to the broader community....

...think. So, if you’ve ever wondered what goes into ‘making virus’ or what gag and pol stand for, we demystify...recombinant adenovirus is missing the endogenous E1 gene, making it replication deficient. This is typically supplied...discussed are compiled for reference below. Happy virus making!  References and Resources Kulanayake, S., Tikoo...

...can begin to understand how the brain works. It’s kind of like “the head bone’s connected to the neck bone... to the rest of the body. Therefore, by simply looking at how neurons are physically connected we can ...efficiently deliver genetic information or tools, making them useful tracers, but finding a virus that works...

... it’s working. You can only do that screen with a system you are really confident about working for most...made standard protocols available and they are working to further optimize those now. They are also developing...

...better fuels, feeds, foods, and fibers. And because working with plants can be challenging, there are a lot...construct our color changing flower we are carefully making our way through the IP landscape, using plant parts...novel pieces where applicable. We will also be working with open source, transformation technologies like...

... and devoted 100% of his energy and effort into making the startup self-sustainable. Within a year from...all safety nets.” Raising seed money. “Before seeking funding, ask yourself if you really want to do ...Co-founder and formerly VP Operations of Sample6) for taking the time to speak with us about the biotech startup...

...inhibition is spatially and temporally ubiquitous, making them a rather blunt instrument for controlling ...and systematically mutated it. Using a robotic picking approach to select cells with variant characteristics...al, Nat Chem Biol 2018. PubMed PMID:29483642. Making protein-protein interaction studies easier with...

...someone speaks about an affinity reagent, they’re talking about a standard antibody: An ~150kD protein with...nanobodies, are derived from a camelid antibody lacking a light chain, called a heavy chain antibody (Muyldermans...clear: it’s an exciting time to be a scientist looking for that perfect fit!  References and Resources...

...between them, could affect the properties or trafficking of the fusion protein (Kimple et al., 2013). ...opsin-expressing cells). For other things to keep in mind when making fluorescent protein fusions, check out Joachim ...Fluorescent Proteins 101: GFP Fusion Proteins – Making the Right Connection.     Thank you to our guest...

...in some applications (e.g. labeling of cells or tracking promotor activity), but it is problematic in applications...for individual cells, and the number of 'normal' looking cells can be counted. This assay will provide information...on the dimerization tendency in living cells. Strikingly, the OSER assay reveals that tagRFP does not ...

...this point, you may be asking yourself, what type of Fab fragment are we talking about here? Many of the...fragment. But this bivalency may cause problems for blocking purposes, since the fragment could bind not only...

...laboratory of Dr. Moshe Szyf at McGill University, working to develop CRISPR-based tools for epigenetic editing...Charlton J, Mattei AL et al (2018). Genome-wide tracking of dCas9-methyltransferase footprints. Nat Commun...Zhong C, Eason DE, Ross DL, Vachhani RV, Ming G-L, King JR, Song H, Sweatt JD (2013) TET1 Controls CNS 5...

...Scientists looking for more compact genome editors due to delivery constraints and scientists seeking a wider... prep in serotype AAV5! Get dLight1.3b here Looking for a knock-out in your experiments? AAV-flex-taCasp3...

...Advancement of Science, Rush Holt, followed by a networking reception. Saturday’s workshop will be an advocacy...laid out by a 2014 National Academy of Sciences working group (National Academy of Sciences. “The Postdoctoral... Advancement of Science, Rush Holt, PhD and a networking reception. Saturday’s workshop will progress ...

... me troubleshoot some technical obstacles when working with virus: 1. Viral vectors undergo DNA rearrangements...cells for infection If your transductions aren’t working, you can check if virus is being generated by your...template. Regardless of the type of virus you’re working with, there’s a lot that goes into virus design...

...recombination events, and that the orientation of the flanking loxP sites directs the Cre recombinase to invert...placement of the two pairs of target sites. By flanking the reporter with one loxP site and one lox511...PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution: The Impact of Site-Specific...

...The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected...amount of virus you actually have in your prep. Taking time to properly titer your virus will not only...original plasmid which can over-inflate results. A packing vector deficient in reverse transcriptase will ...

...or the activity of a protein in a synapse. What kinds of tools enable us to measure these properties, ...articles describing the FRET tool that you are looking for and check our curated list of biosensors too... Sustarsic, Marko, and Kapanidis, Achillefs N. "Taking the ruler to the jungle: Single-molecule FRET for...

...alleninstitute.org. A classic challenge in cell biology is making sure that what we observe through the microscope...whole segment—a GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into...and destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag...

...density: particle concentration is measured by looking at absorbance at 260nm using UV/Vis spectrophotometry.... The purity of the sample can be estimated by looking at the ration of DNA (260nm) versus protein (280nm...calculated in terms of plaque forming units (pfu), taking initial vector dilution into account. You'...

...10.1038/s41587-023-01756-1. PMID: 37095348. Breaking up is hard to do: Monomeric StayGold variants ...with some applications. Several labs have been working to produce monomeric versions that break up the...was delivered to brain endothelial cells in mice lacking LY6A using AAV1-X1 (Chen et al., 2023); then, when...

... who keep changing their minds and have trouble making decisions. In my work as a thesis coach for graduate...categories. In addition, I also learned about two other kinds of difficult supervisors: Hands-off super busy ...explain how it is interfering with your work. Avoid talking about your emotions, such as frustration or anger...

... I’m not going to lie, AAV transfer plasmids are kind of a pain to work with. These plasmids contain ITRs...the viral particles of the AAV serotype you’re working with are primarily found in the culture media, .... If you’re new to creating gradients, practice making the iodixanol layers before doing your first purification...

...in brackets and add the upstream and downstream flanking sequences to their input string (Figure 2). This...prediction models. In addition, we provide options for nicking gRNAs (used for PE3 and PE5 prime editing strategies...strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019). PRIDICT saves time and resources by ...

...components of the indicator, which can make the kinetics of voltage sensing to signal output slightly slower...fluorescent signaler. The pro of this system is the kinetic response time from signal input to readable output...FP-rhodopsin GEVIs have been engineered to have kinetics comparable to its one component counterpart and...

... localization, cell structure, intracellular trafficking, and protein turnover rates. Additionally, by...fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery of many ...cellular mechanisms, there are some limitations to working with FPs. Overexpression of fluorescently tagged...

...the FP (fused to the protein of interest), thus tracking the localization of that biomolecule inside living...eel was shown to mature independently of oxygen, making suitble for use in anaerobic conditions. Maturation...fluorescent protein should I use?" And if you're looking for more fluorescence microscopy tools and techniques...

...cellular functions, including ubiquitinoylation, kinase signaling, integrin signaling, and transcription...Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection. Cellular expression Whether...localization may differ from those of your BiFC construct, making it difficult to interpret your results. Unfortunately...

...our samples 1:6 million to 1:25 million.  After making the dilutions, they are transferred to a new plate...performing ddPCR Clean everything You must have a clean working area to titer your AAV by ddPCR. Droplet digital...generator, you should avoid pipetting too quickly when making your virus dilutions. This will reduce aerosols...

...dye, mauvine, was discovered in 1856 by William Perkin. His discovery set off a flurry of activity and...labeling strategies and improved fluorophores are making chemical dyes increasingly attractive and accessible...Medical Institute. He would rather be in the lab making new fluorophores for advanced imaging modalities...

...domain proteins, they can be expressed in bacteria, making nanobodies a recombinant and renewable anti-IgG...available nanobodies from Pleiner et al. Before picking a secondary nanobody, it’s important to review ...expression plasmids here Additional Resources Looking for monoclonal primary antibodies to use with the...

...does not do). Furthermore, molecular biologists working with vectors are, in most cases, interested in ... the viral integrase protein (IN) during step 3 (King et al., 2008) (Hu & Temin, 1990). At step 3, the....1016/0022-2836(73)90277-5. PubMed PMID: 4355596 King, S. R., Duggal, N. K., Ndongmo, C. B., Pacut, C....

...zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis... target mRNA inhibit its translation either by blocking initiation or splicing. (RNAi, in contrast, engages...unprecedented views into cognitive processes like decision making (Kim et al, 2017). “Brainbow” fish lines have been...

... delivery to only a few cell types.  If you’re looking for more control in the cell types your lentiviral...retrograde direction from the synapse to the cell body, making it a great tool to study neuronal connectivity....HY, Tortorici MA, Veesler D, Murphy M, Pettie D, King NP, Balazs AB, Bloom JD (2020) Protocol and Reagents...

...a bit of tuning before you know your system is working as expected. Finally, for each gene you want to... scorer 1.0 (Now 2.0) (Chari et al., 2015) and picking whichever guides are closest to the transcription...Ansari R, Harper S, Jackson DA, McRae R, Pooley R, Wilkinson P, van der Meer D, Dow D, Buser-Doepner C, Bertotti...

...that particular region. No matter where you are looking, you can either scan the genome yourself for potential...leading to degradation that can set you back days. Working with plasmid DNA is much easier and doesn’t require...slightly different method to genotype their pups. Taking a toe, a finger, a tail tip, or a combination of...

... of how they actually “report” as biosensors. Making aptamers glow The flexibility of aptamers means...fluorogen and increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required... Eric J. Perkins...

...BP Reaction takes place between the attB sites flanking the insert and the attP sites of the donor vector...gene! STEP 2: Generate an Expression Clone When making the expression clone, it is important to choose...fragments, cDNA or Genomic DNA and is available for all kind of organisms from mammals to E. coli. Glossary of...

...reliable and robust high quality viral vector service, making an array of ready-to-use viral vectors available.... I would be remiss to talk about them without looking at what they’re delivering and where they’re delivering...of our most popular ready-to-use viral vectors, making up almost 10% of our requests by 2019 (Figure 5A...

... replication and regulation in bacteria Before looking at an example plasmid, let's review how E. coli...different ways: methylating newly synthesized DNA, blocking binding sites, or trading out ADP for ATP to re-activate...mRNA and prevent this pseudoknot from folding, blocking replication. Created with BioRender.com. Plasmids...