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... control sample. Southern blotting applications range from confirming the results of cloning or knockout...days of development. Today, researchers use both Sanger and next-generation sequencing techniques to answer...see below). Importantly, although their role has changed, restriction enzymes are certainly not dead yet...next-generation sequencing techniques Introduced in 1977, Sanger sequencing, also known as chain-terminating or ...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides... and accessibility led to the widespread use of Sanger sequencing. Building upon this technology, radiolabelled... sequencing techniques do not typically rely on Sanger sequencing but instead use methods like pyrosequencing...

...reality – a government cannot declare that gravity change direction and make things fly up when we drop them...outcomes are actually possible for a given policy. In exchange, the government protects (and often funds [3])...scientists has been rising as evidence of climate change, the safety of genetically modified organisms (...90% of active research scientists believe climate change is mostly due to human activity while much less...administration’s actions, including taking down the climate change page on whitehouse.gov, freezing grants, and proposing... Agency, and the National Institutes of Health endanger scientists’ ability to uphold their side of the...advocating for science education and literacy that this changes.  2) Lumping all scientists (and consequently their...

...various teal, green, yellow, orange, and red fluorophores. The spectra for mOrange2 (scaffold of OCaMP) are...signaling throughout the body. Because calcium levels change rapidly during neuronal activity, scientists can...are shaded in orange. Data from FPbase.org. Scientists have developed calcium indicators spanning ...complement these tools, we recently developed a new orange calcium indicator, OCaMP (Aggarwal et al., 2025...2025), based on the fluorescent protein mOrange2 (Figure 2). Figure 2: OCaMP structure and mechanism...CC-BY-NC-ND 4.0 International license. Why orange? OCaMP’s emission peak at 565 nm fits neatly between...for two-photon imaging with newer lasers in the range of 1000–1080 nm. These industrial fiber lasers are...

...and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved .... Cell Rep. 2015. PubMed PMID: 26190106 PRESTO-TANGO: An open-source resource for drug discovery Nearly.... Bryan Roth's lab has a newly available PRESTO-TANGO kit which allows researchers to interrogate the ...publication, Kroeze et al. describe their enhanced TANGO arrestin recruitment assay incorporating 314 codon-optimized...cell line expression. In a twist on the classical TANGO assay, Kroeze et al. developed a new approach termed...discovery. Researchers can use plasmids from the PRESTO-TANGO kit directly for drug screening or easily shuttle... more rapid screening by restriction digest and Sanger sequencing, respectively. The speed and simplicity...

...outside of their natural environment. Thus, the Harris Wang lab at Columbia University developed a method for...both Gram-positive and Gram-negative cells.  The Wang lab further developed a modular suite of plasmids...replicative origins that can be used for broad or narrow range of microbial species. These also contain the RP4... RSF1010 origin, an IncQ origin with broad host-range (Meyer, 2009). The vector retains the oriT for delivery... by conjugation. The labs screened for the host-range of the plasmids and suitability of various antibiotic...designed to study the bee microbiome, these broad-host-range plasmid could also be used to study other microbiome... the toolkit for modular assembly of broad host range plasmids Counting cell division in the gut microbiota...

...register and catalyzes their recombination, thereby changing the state of the RSM. If an attB-attP pair is ...orthogonal attB-attP pairs per recombinase may be arranged on a register (Fig. 1C below). If attB-attP pairs...approach is to interrogate state with qPCR. The re-arrangement of DNA segments in each state of a register enables...methods of state interrogation mentioned above (Sanger sequencing, qPCR, and fluorescent reporter-based...each come with their advantages and disadvantages. Sanger sequencing is simple and the most accurate method...directly reads the state of a register. However, each Sanger sequencing reaction can only be performed on one...or if it requires reading so many registers that Sanger sequencing would be too expensive, then the qPCR...

...Wei Y, Liu F, Ying W, Zhang J, Tang C, Zhang X, Xu H, Shi L, Cheng L, Huang P, Yang H (2018) In vivo simultaneous...experiment! References Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable...for each loci, CRISPR/Cas systems only require changing the programmable guide RNA. Gene activation by... histone acetyltransferase domain capable of rearranging chromatin structure. This method has shown higher... Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation...

...physiological conditions. Shyu et al. Biotechniques. 2006 Chang-Deng Hu Green sfGFP Split super-folder GFP...fluorescent protein. Kamiyama et al. Nat Commun. 2016 Bo Huang spGFP Split superpositive GFP Split-superpositive...protein labeling. Feng et al. Nat Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1–10) and mNG3A(1–10) for ...signal amplification. Zhou et al. PLoS One. 2020 Bo Huang Yellow Venus pBiFC-VN173, pBiFC-VC155 Identification...physiological conditions. Shyu et al. Biotechniques. 2006 Chang-Deng Hu Venus Improved N-terminal fragment VN155...-noise ratio. Kodama et al. Biotechniques. 2010 Chang-Deng Hu mVenus pET-BiFC contains both fragments...protein labeling. Feng et al. Nat Commun. 2017 Bo Huang sfCherry3 Improved sfCherry3C(1–10) for use ...

...product no one has ever seen before (flowers that change color throughout the day) and doing this with very...our lives and took this daring risk? What moment changed our whole outlook on the possibilities in life ...published a paper a year (or less) and limited the exchange of information to the public and to other academic...fair game for speculation - in fact, our color changing flower project was inspired by a Facebook comment...public is can be fearful of this technology and angry at the agriculture companies that produce GMO food...the detector plants, we expected to see that same anger. Instead, social media comments were more often ...we hit upon the idea of building a flower that changed color throughout the day. It would be a beautiful...

...chromoproteins are just now being developed for a broad range of biological inquiries.  What is a chromoprotein... plasmids (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue...specific purposes.     Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP... create negative selection markers for allelic exchange. These vectors contain a toxin gene (regulated...rhamnose-inducible promoter) and a chromoprotein gene. Allelic exchange occurs through two sequential homologous recombination... the tsPurple chromoprotein.  Find the allelic exchange vectors here! ShadowR, a novel FLIM-FRET acceptor...Image: Murakoshi et al., 2019. ShadowR is an orange light-absorbing chromoprotein that Hideji Murakoshi...

...s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and easily...screening. They and their colleagues including Feng Zhang used the GeCKO library to identify genes essential...lentiviral CRISPR libraries developed by MIT's Tim Wang and colleagues, including David Sabatini and Eric...limited to modification of a few loci at a time,” Wang said. “It was very difficult to screen in a pooled...targets at once. That’s what our plasmids allow.” Wang’s library is designed to target a smaller set of ...titer due to the smaller size of the plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single...target any sequence in the genome. Tips and tricks Wang offers this tip: “It's quite important, at every...

...fused to the N-terminus of LSSmOrange pLSSmOrange-C1 Orange Mammalian Express a gene of interest fused...GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest fused... (the acceptor) by means of intermolecular long-range dipole-dipole coupling." The emission of the acceptor...FRET has also been used to measure conformational changes, cleavage activity using FRET-based biosensors,...fused to the C-terminus of LSSmOrange mRuby2 Red Mammalian Expresses mRuby2 (a RFP variant) commonly used...method useful for dynamic experiments in which FRET changes are large. Two other methods are commonly used... such as pH or autofluorescence background, can change the fluorescence decay time and have to be taken...

...mNeonGreen can be used as a fusion tag for a wide range of applications from traditional imaging to FRET...from the coral Clavularia sp. protein CFP484. Hui-wang Ai and colleagues from the University of Alberta...mTFP1 can be used as a FRET donor to mCitrine and mOrange or for live cell imaging of fusions and FRET reporters...is also insensitive to physiologically relevant changes in pH. Find mTFP1 plasmids here! mWasabi: a bright...mutagenesis approach and found that specific amino acid changes favored blue-shifting more than others. During ...particularly with fluorophores that excite in the violet range. For example, mWasabi can be used with Sapphire ...deposit the plasmids at Addgene! References Ai, Hui-wang, et al. "Directed evolution of a monomeric, bright...

...body monomers respond to pH changes by undergoing local conformational changes. These tiny movements are... much force can be generated by conformational changes in proteins? How can information propagate through... repeatedly extend and contract in response to changing pH, and they can rupture E. coli spheroplasts ...and retract over many microns in response to pH changes without the need for ATP or other chemical fuel..., R bodies do not exert force by dynamically rearranging into different polymerization states. Instead...vasculature or extracellular matrices) in response to pH changes. Beyond these applications, purified R bodies ...

...sufficient to prevent any pH change, as we still observed a slight change in pH (from 7.4 to 6.8). It’...tubes (Fig. 1). It’s important to note that the change in pH only occurred when the solution thawed, as...samples were frozen. This was clear by the gradual change in pH visible at the sample interface upon thaw...found that 30 seconds of venting attenuated the pH change caused by storage on dry ice (Fig. 3). However,...CO2 to leave the tubes, and would prevent the pH change upon thaw (Fig.4). To test this, we froze the tubes...stability (Murphy et al., 2013). However, the pH changes in our solutions are transient and return to normal... have no evidence to suggest that the transient change in pH affects AAV function. Furthermore, another...

... other CRISPR plasmids are from the Zhang lab. In fact, the Zhang lab has shared over 40,000 CRISPR-Cas9... different cell types so it is useful in a wide range of applications. This plasmid was the top requested...pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses Cas9 from Streptococcus pyogenes with...generation lentiviral transfer vector also from the Zhang lab is used to deliver Cas9 and your gRNA of interest...genome engineering. For use in human cell lines, the Zhang lab codon-optimized SpCas9. Using the BbsI restriction...BB)-2A-Puro (PX459) V2.0 - This plasmid from the Zhang lab is a mammalian vector that expresses both SpCas9...original publication but its name was subsequently changed to clarify that it does not contain the trademarked...

...into one of those unexplored areas with our color-changing flower, but to do that we’ve had to navigate the...modification (GM) and synthetic biology. The mistrust and anger directed at GM crops has burdened this emerging ...product specifically for consumer sales. Flowers that change color throughout the day cannot be developed through...of basic research standing behind it. Our color changing flower involves two basic components: the pigments...combine these components to develop flowers that change color. Without this basic research – this comprehensive...sometimes have limited distribution. We hope that will change with Addgene’s expansion into plant synthetic biology... GreenGate, and we are excited to see how this exchange of genetic tools facilitates plant science. Challenge...

...your expected range. For example, calcium in the ER lumen reaches the sub-millimolar range, much higher...calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent change in fluorescence only occurs...systems tend to have slower, but larger, calcium changes. The GCaMP6 family of sensors are commonly used...compartments can vary greatly. Additionally, the calcium changes you will observe in your experiment could be subtle...are available to simultaneously measure calcium changes in the ER, cytosol and mitochondria), the Cameleon-based...ratiometric GECO sensor (GEX), where one wavelength changes with Ca while another wavelength is insensitive...sensors have also been re-engineered to measure changes in calcium specific to the neuronal cell body or...

... positive change in the scientific enterprise and the way it is funded and to learn tangible skills necessary...necessary for affecting change. On Friday, the symposium includes a panel discussion and keynote by former...the Advocating for Science Symposium Boston 2016 Changes since the Future of Research Symposia Ongoing ...initiated. Recent developments promise to further change the landscape facing all scientists, both young...available avenue. Interestingly, both of these changes share a common thread that has the potential to...for the future No matter your opinion on these changes, it is likely that the shifts caused to the practical...example, implementation and compliance for the FLSA changes, including finding money in already tight lab budgets...

...donor-acceptor pair and a change in FRET efficiency correlates with a change in the distance and/or orientation...fluorescent protein (i.e. rearranging the start and end positions without changing the order of the amino...to a change in the environment which alters the FRET efficiency. Detecting certain cellular changes with...biological processes occur within the typical FRET range, FRET efficiency is used to infer an interaction... protein based FRET pairs with improved dynamic range for fluorescence lifetime measurements." PloS one...studying protein-protein interactions by measuring the change in FRET efficiency, from which conclusions regarding...