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...used to calculate the concentration of the sample. This concentration can then be used to calculate the viral...generator to the PCR plate. Maintaining a high droplet count is important for calculating the concentration...Addgene, we typically load 3 dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall...making the dilutions, they are transferred to a new plate containing the mastermix which includes primers ...a droplet reader extracts the droplets from the plate and measures the fluorescence amplitude of each ...positive (blue) and negative (gray) droplets. The no template control wells should have very few positive droplets...approximately 1 positive copy per microliter in the no template control wells. You’ll also notice a reduction ...

...are sigmoidal. Once the trendline is calculated, you can extrapolate the concentration of the unknown sample...software to calculate the data’s coefficient of determination, R2. R2 is the square of the correlation between...could be because there is no correlation or it could mean that the relationship is non-linear and you should...versus the response and use that information to extrapolate the concentration of an unknown sample. For example...the accuracy of the curve, which we will discuss later.       Standard curves must be included every time... the experiment and use the standard curve to calculate its experimental value. Though it varies from ...measurements and the trendline y = 0.6561x + 0.019 calculated. The concentration of an unknown sample is determined...

...antitoxin; Modulation of killing (mok), required for hok translation. Here, Mok and Hok translation is coupled...that is regulated by antisense RNA, which base pairs to cognate mRNA to inhibit its translation. The hok...coupled. Translation from the Shine-Dalgarno (SD) sequence of mok results in translation of Mok and Hok...restriction-modification systems onto plasmids allowed cell populations to retain more of these plasmids as compared ...the cell grows resulting in the appearance of unmethylated sites in the chromosome. The more stable endonuclease...chromosome leading to cell death. Antisense RNA-regulated systems Toxin-antitoxin systems are not just restricted...hok/sok system is the first antisense RNA-regulated toxin-antitoxin system discovered. Unlike the systems...

...detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published... to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar... to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How does... for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly...reliably distinguish between Zika and a closely-related virus, Dengue from multiple sample sources. SHERLOCK...from cell-free DNA fragments as well as health-related single nucleotide polymorphisms (SNPs) from human...

...we shared materials and equipment between labs. Later I worked with people outside the department to accomplish... connect the imaging community and to create a platform for discussions and potential collaborations. ...loads of opportunities to create communities, platforms, and to be a part of projects that connect people...realize. Being at a university provides the perfect platform for practicing initiative and for training in ...conference calls and make sure to maintain these relationships by keeping in touch by email and social media...media (see the section on “Maintaining Relationships” below). Expand: Collaboration opportunities outside... fun. I just had to remember to approach the relationship with the perspective of my potential collaborators...

...antigen present in the sample and is calculated by extrapolating from the standard curve. Because the ...antigen present in the sample and is calculated by extrapolating from the standard curve. Scientists often...same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent may ...of the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A...substrates since they can be read on a standard plate reader as opposed to chemifluorescent, and chemiluminescent...antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody first binds...primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody that binds...

...undergo significant post-translational modifications including glycosylation, disulfide bond formation...proteins functionally relative to the untagged protein of interest. To avoid the accumulation of dark fluorescent...is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent... fold? Misfolded FPs do not fluoresce. A dark population is not readily apparent and can confound quantitative...construct [1,2], i.e. positioning of the FP sequence relative to cell compartment targeting sequences, which...1) [4,6–8]. Additionally, FPs that encode N-glycosylation consensus sequences are modified with the addition...addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...

...How are inducible promoters regulated? Inducible promoters can be regulated by positive or negative control...Addgene! Chemically inducible promoters Chemically regulated promoters are among the most common inducible ...protein purification. When arabinose is absent, regulatory protein AraC binds O and I1 sites upstream of...complexed with cAMP activator protein (CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing...include positive inducible alcohol and steroid regulated promoters commonly used in plant research. ...Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA promoter AlcR pGGA008 (AlcA) and pGGC011...AlcR) from red flame GreenGate kit Steroid-regulated LexA promoter XVE (synthetic) pFZ19 (yellow flame...

...externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists can make...make it easy to study the effects of genetic manipulations. In this blog post, we’ll cover five popular... model organism for scientists as they have a relatively short generation time for mammals - the time ...study diseases such as cancer since mice better recapitulate the complex interactions between cancer cells...are genetically tractable. Mice can be easily manipulated with tools like CRISPR to make transgenic lines...entirely sequenced and is very amenable to genetic manipulation. Yeast cells are great model organism not only...studied in other model organisms that better recapitulate in vivo conditions similar to humans. For example...

...enhance or upregulate ncRNA expression. This approach is useful for lncRNAs because they regulate nearby ...They play important roles in many cellular and regulatory processes, but much of their functionality is...unveiled as they are more difficult to functionally manipulate and study in comparison to protein-coding genes...to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe the effects. Luckily for...of disrupting adjacent genes, especially those regulated by or overlapping with the lncRNA (Basak and Nithin...effects can be minimized by disrupting lncRNA regulatory elements that are crucial for transcription and...and Bester 2021). For example, inserting a polyadenylation signal downstream of the transcription start...

...membrane-impermeable biotin substrate (Figure 1). Biotinylated proteins were isolated, identified by mass spectrometry,...incubators, micropipettes, and plate readers. Instead, students can use inoculation loops for liquid transfer...as AAV Packaged on Request) will be useful for related studies in other brain regions, cell types, and...enabling better spatial resolution for neuronal populations in vivo in both mouse and zebrafish brains. ...photometry. Plus, both designs offer promising templates for future improvements. Find PinkyCaMP plasmids...calcium indicator for in vivo imaging of neuronal populations at single-cell resolution. PLoS Biol. 23(4): ...fight cancer. In this method, called adipose manipulation transplantation (AMT), CRISPR-modified adipocytes...

...antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target protein is added...coronaviruses into a llama over several days and isolated special camelid antibodies that bind to the spike...secondary nanobodies blog post.  A rapid selection platform for generating sybodies against membrane proteins...weeks (Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly...in the form of mRNA for academic research. The template plasmids for generating your own libraries are...Generation Toolbox! Starting with libraries and a biotinylated (for purification purposes) protein target of...involves using a commercial kit for in-vitro translation of the mRNA libraries. Each mRNA molecule in ...

...gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness...could eventually spread through the entire wild population with unpredictable ecological effects. Because...chromosome and its own gene is used as a repair template, thereby converting the heterozygote into a homozygote...building gene drives that can spread through populations of transgenic laboratory organisms but not wild...sgRNA-only drive will spread exclusively through populations that already express Cas9 from an unlinked locus...sequence will only spread in transgenic laboratory populations with that sequence2. Both methods are easy to...performed in regions that don't have resident populations of the relevant mosquito species. Reproductive...

...expression, and protein translation. Its genome consists of “early” (E) and “late” genes, and the transition...transition between early and late infection (and therefore between early and late gene transcription) is demarcated...unusual cases of acute hepatitis in the pediatric population. Once ~400 cases had amassed in places around...exist. The life cycle of AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of ...recombinant sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid...expressed, since the early genes are absent, and the late genes (and their structural proteins, which would... some of the wild-type viruses can persist as a latent infection in certain tissue reservoirs (Pereira...

...optogenetic stimulations, they simultaneously recorded the electrical impulses of discrete subpopulations of this... or reward-related behaviors. The high spatio-temporal precision and reversible modulation of FLEx vectors...combined optical and genetic methods to photo-stimulate mammalian neurons and alter synaptic events: a..., 2005; Deisseroth et al., 2006). Despite its relatively recent development, the history of optogenetics...rhodopsins and neurons have in common? Neuronal stimulations are triggered by the movement of ions across...Zhang, 2012; Towne et al., 2016). Optogenetic neuromodulation is made possible by the light: when hit with...viral-infected neurons are subsequently photo-stimulated through fiberoptic cannulas directly implanted...

...is its amenability to genetic manipulation. Tools for gene manipulation together with information about...drive biology is to examine the consequences of manipulating genes. One reason for the success of zebrafish...groundwork for understanding the gene networks that regulate processes from development to metabolism to fertility...back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated in congenital...morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. (RNAi...(RNAi, in contrast, engages existing gene regulatory mechanisms to block activity and does not work easily...compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). Generating zebrafish...

... build the relationship in a non-awkward way?  7 ways to strengthen your new relationships Here are some...Relationships need nurturing. Perhaps you’ve met someone interesting at an event. Or, perhaps you have...contacts you have; it’s about building genuine relationships with people you care about knowing. How do you...some ways to go about strengthening a new relationship with authenticity and not discomfort: Invite the...their new publications via Pubmed, so you can congratulate them when there’s good news.  Send an email ...meeting (in person or virtually) will make the relationship stronger.  If appropriate, invite them to speak...department. Even if it doesn’t work out, it’s flattering to ask.  Show appreciation. Send a thank you ...

...customized to control transgene expression. Gene regulatory elements [promoters and enhancers, miRNA target...IV injections are typically administered in the lateral tail vein or retro-orbital sinus. IT injections... are “seeing” a great number of cells as they circulate in the blood, rather than being geographically...Abbreviations: AAV, adeno-associated virus; GRE, gene regulatory element; NHP, non-human primate. Many of...., 2023 AAV-PHP.V1* CNS: Biased toward vasculature, but also transduces astrocytes Mice Yes ...al., 2020 AAV-PHP.V2 CNS: Biased toward vasculature, but also transduces astrocytes Mice Yes ...(rats, infant green monkey, rhesus macaque); vasculature (adult marmosets), biased toward astrocytes (...

...Listen to the podcast segment on modulating iPSC reprogramming  Regulation of telomere length with TZAP .... Subscribe to the Addgene blog to get all the latest hot plasmid updates!     Tol2 Gateway-compatiable... 2015. PubMed PMID: 25772473 Targeting and manipulating interneurons using a viral approach Genetically...’s lab has developed a new AAV approach using regulatory elements that restrict expression to this cell...approach allows you to target and functionally manipulate GABA-ergic interneurons across species and will...combination of deliberate and random mutagenesis to isolate the best RFP. They targeted known residues on the...brightness, and proper maturation. Three new RFPs were isolated and characterized: mScarlet (fluorescent lifetime...

...proteins that promote RNA decay and their upregulation is correlated with poor survival in breast cancer patients.... BirA* requires 16-18 hours to sufficiently biotinylate interacting proteins, which results in poor spatiotemporal...interactions with RaPID RaPID is similar to the BioID platform for identifying protein-protein interactions, ...promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that are within ~10 nm of the bait, ... also tweaked BirA* to increase its rate of biotinylation. The improved enzyme, called BASU, is derived...one to study RNAs expressed from their native regulatory elements. Controls for RaPID experiments RaPID...Affinity Purification (CRAPome), to identify highly biotinylated background proteins reported by other research...