...Mechanisms of epigenetic modifications The main mechanism of epigenetic modification is chromatin remodeling...literally means ‘on top of the genome’). Epigenetic modifications can result in phenotypic changes without any...any change in genotype. Epigenetic modifications play a large role in turning genes ‘off’ and ‘on’, signaling...two main mechanisms, either through histone modifications, which can influence the transcription of genes...hypomethylation, on the other hand, has also been implicated in the development and progression of cancer ...enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. However none of ...can be used for in-situ research of dynamic modifications in real-time, posing a huge challenge to studying...

...There are several important considerations with fabrication of the fiber optics needed for optogenetics. ... for the implant depends on the dorsal/ventral location of your region of stimulation (the z-axis bregma...emitted from the end of the fiber optic implant scatters a great deal once it enters tissue, placement ...0.5mm above the site of your stimulation. The scatter effect is also an important consideration when ...Lasers can be purchased through Laserglow, which caters to optogenetics users and adjusts the lasers appropriately...require stimulation only under certain contexts/locations in the test apparatus and thus individual laser...inactivation kinetics. For example, the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several...

...entire page dedicated to different career options from science policy to science communication and much ...), about their experiences. Research requires dedication and effort, so building a strong understanding...critically about whether it’s something you want to dedicate your time and energy to. How can I make the most... feel less confident in their abilities and qualifications. Studies on imposter syndrome have also found...universities often have scholarships and funds dedicated for undergraduates interested in doing research...the experience would improve my scientific and application writing skills, and ended up with the award. ...fantastic for broadening your experiences! These applications can be highly competitive, so asking for help...

...for successful modifications, efficiency can be low, especially for larger modifications. What’s the solution...several potential CRISPR applications. First, CRISPR can be used to target locations in the genomes of pathogenic...tools available for bacteria and some of the applications for which they’ve been used. The beginnings ... of Enterobacteriaceae, without the need for modification. This finding suggests that the system may be...eukaryotes (Qi et al., 2013). As in other systems, catalytically dead (dCas9) targeted to a promoter or gene ...responsive promoter to drive expression of a catalytically dead Cas9 enzyme allows reversible repression...Bikard et al., 2013). By targeting either a catalytically dead Cas9 (dCas9) alone or a dCas9 fused to ...

...many CRISPR applications, and they’ve also had success in clinical gene therapy applications. Read on to...to learn more about the current (and future) applications of lentiviral vectors! Lentiviral vector history...original system, the possibility of creating a replication-competent virus via recombination between the...of recombination events necessary to create a replication-competent virus. Second, Tat is eliminated through...barcoding libraries have been used for complex applications like lineage tracing in heterogeneous tumors...2015: lentiCRISPR v2, used both for targeted modification and genome-wide screening.   Lentiviral vectors...developed integrase-deficient lentiviral vectors for applications that require the large genetic capacity of a...

...manuscript to get reviewed and edited prior to publication, and this will delay the dissemination of your...deposited by George Church’s lab). Addgene has also cataloged a variety of useful molecular cloning protocols...bacterial transformation. And more plasmid-related educational resources can be found in our Plasmids 101 blog...bring your phone into the lab? Maybe having a dedicated lab-use tablet? The software also makes it easy...for biologists and bioengineers to share their educational materials, protocols, and courses, as well as...equipment, and track inventory and orders. Quartzy caters to life science labs and offers the service free...scientists by allowing companies to pay publish their catalogs on the site. Like Labguru, Quartzy offers software...

...emission wavelengths, which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These... substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...typically bind to. Stability of fluorescence: For applications that involve ranges of pH, and significant lengths...light, is another factor to consider. If your application requires longer periods of exposure, you’ll want...degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic nature. The potency and efficacy...descriptions, or technical manuals. Specific applications and matching dye/ligand choices For effective...

... 2023 * indicates viral preps available. Mechanisms of crossing the BBB are indicated in parentheses...suitability of a systemic capsid for an individual application is dependent on a number of factors, including...test your chosen capsid before using it in an application.     Figure 1: AAV toolkit for targeted...doses are recommended for all systemic capsid applications. For example, AAV-PHP.eB is typically administered...experimentally determine the best titer for your application.   Pro tip!Want to know if a capsid is a good... capsid immunogenicity is a concern, using a purification method that removes, not just reduces, empty...always, validate the capsid for your specific application and animal model.  Other model organisms Tropism...

...done using side scatter (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell... the plot that divides it into four, using the location of the cell populations as a guide  (Figure 3....be used for this purpose.  Pro tip! The forward scatter is a proxy for diameter and volume of the cell,...the exclusion of doublets and debris. The side scatter roughly measures granularity, a proxy for nucleic... will have numbers by gated populations; this indicates the percentage of total events contained in the... gated population. Heat maps are often used to indicate the relative density of cells. You can see both...presented. A horizontal line across the peak will indicate the cut-off value for a positive signal (Figure...

...Figure 1: Choosing the best PE6 variant for your application. Image reused form Doman and Pandey et al., 2023... CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features of DIRECTED...approach for experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D...delivery by modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467...interactions or map a local proteome, but some applications have been challenging or inaccessible. The Ting... (which had therefore been trafficked between locations). Figure 3: A) LOV-Turbo becomes active...active when illuminated with blue light, catalyzing the biotinylation of nearby proteins. B) TransitID uses...

...be part of the answer. Problems with written publications Online tools like ResearchGate, GoogleScholar... that the big breakthroughs in my discipline, education, will come from ideas garnered from research in...from the science of others. Improving science communication through video     Video abstracts about research... to be that video is the future in terms of communicating ideas. In 2012, for example, 3 billion hours...scientific videos were for entertainment or basic education (i.e., learn why water freezes) there was a growing...video usurps the written word as the means for communicating research. Short videos of 3 to 5 minutes provide... researchers to leverage the video medium to communicate their research more effectively.” How to share...

...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysS...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysE...Expression vector cannot have p15A origin of replication F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (...Expression vector cannot have p15A origin of replication fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS/ pLemo...Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15, thi, lac-, mtl-, recA+, KmR Expression... commercial strains carry the DE3 lysogen, as indicated by the name of the strain. Conversely, other strains... for positive selection and a p15A origin of replication, making it incompatible with other p15A plasmids...

...Addgene ID number, your publication will be listed on our website as a “Publication Citing this Plasmid” ...upon other researchers being able to find their publications in the first place. As a curator of the thousands...make it easier for you to find the plasmids and publications relevant to your research interests. We do this... this by promoting citation, supporting sophisticated search, and curating plasmid collections. Addgene's...number as well as the reference for its original publication. To help facilitate this process, we explain ...fruits of your labor and won’t have to repeat the modification. This will save both time and money. A quick...whether the plasmid has been used in your system. Sophisticated search You can search for specific plasmids ...

... use the GAL4-UAS system along with precisely truncated attP sites to control recombination of the gene...of PhiC31. In the SPARC system, attP sites are located on either side of a stop cassette, upstream of ...amount of labeling, you can alter the attP site. Truncating the attP site upstream of the stop cassette results...population is directly correlated to the length of the truncated attP site. The three SPARC variants, based on ...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...Image from Isaacman-Beck et al., 2019. Applications for SPARC SPARC technology is versatile and can be ...dendrites of T5 neurons by placing the calcium indicator GCaMP6f in the effector position, as well as to...

...well as protease sites to facilitate protein purification. We hope this kit will allow you to quickly ...for a variety of synthetic biology or protein purification studies in E. coli. 800 and 1900 Series If you...polycistronic E. coli expression constructs. Some applications of plasmids from this kit include the production...and Charles E. Melançon III. "Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics...designed the pCri system to simplify the protein purification process by making it easy for a researcher to... blog post on the system Easing the Protein Purification Process with pCri Cell-free protein expression...Daniel, et al. "A pull-down procedure for the identification of unknown GEFs for small GTPases." Small GTPases...

...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...steric and charge-based nature of the phosphoryl-modification; they are both much smaller, and carry only ...thus cannot reflect the true state of protein modification within the context of its appropriate regulation...codons (TAG), replacing them with TAA (7). This modification allowed the deletion of release factor 1, the...optimize protein production using standard protein purification strategies; expression should be optimized for...-incorporated to non-phosphorylated protein. Verification of site-specific pSer incorporation can also...programmable protein phosphorylation." Nature communications 6 (2015). PubMed PMID: 26350500. PubMed Central...

...study its function is one of the most popular applications of fluorescent proteins. These fusion proteins...steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding sites or motifs that...that are necessary for post-translational modifications. Despite these potential issues, GFP has been used...linker sequence short In the early days of GFP applications, many were concerned with steric hindrance by...N-terminus of the donor fluorescent protein can be truncated to increase the FRET efficiency. Creating fusions...fluorescent protein and protein of interest (POI). To indicate the sequence, people often use terminology like...  Determining other aspects of functionality (catalytic activity or interactions) can be challenging. ...

... This allows them to replicate viral plasmids with the SV40 origin of replication. To generate a virus...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first...cell biology, there are thankfully many other applications that they are useful for! What the HEK is it...cell line and we will highlight some of those applications here.   Virus production HEKs are the go-to ...proteins at high levels. In a case where clinical applications of recombinant proteins are necessary, HEKs ...significantly reduces the cost of production. Research applications There are two areas of research where HEK cells...vulnerable to viral infection. Yes, your cells can catch a cold—or worse! HEK-293T and its derivatives are...

...  After identification of ABE activity, XMAS-TREE can also be used to purify these modifications and work...ABE-targeted cells By Ellie Adams Single base modifications through ABE or CBE's have the potential ability...point mutations. They allow single-nucleotide modifications to edit DNA without requiring a double-stranded...difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells are...mCherry and GFP expression to check for ABE’s. b) Quantification of base-editing efficiency using XMAS-TREE-...methods at various genomic loci in four different categories.     Brookhouser, N., et al. BMC Biol 18...MYC-dependent cancer cell lines. It can be applied for identification of essential MYC binding sites in a wide range...

...Scientific Publication, where diverse approaches to peer review were discussed. Science communication opportunities...developmentally programmed regulation of DNA replication and gene transcription during vertebrate embryogenesis...There is a rich and active community of science communication professionals who are thinking about, for example...access affects financial models in information publication. Take a look at the blog ‘Scholarly Kitchen’ ...opportunities If you are interested in the science communications world, you might consider an internship or ... is enjoys broad engagement through science communication with a variety of audiences. She focuses at ... science communication...

...protein-only transduction method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only...the HIV protein Tat. Although CPP fusions can translocate a cell membrane, the presence of the CPP may ...that components in the buffer used for protein purification must be helping the untagged protein enter cells...Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP using Cre-lox recombination...showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency of biallelic mutations. Importantly...Importantly, hESCs retained their stemness, indicating that iTOP may be valuable for CRISPR/Cas9 stem cell...potential for off-target effects. Third, protein purification (once a protocol has been established) may be...

...Gibco organizes its app into four categories: tools, analytics, education and data manager. The tools are...are five FREE science lab apps that have been educational and great for lab life. These apps prevent you...lab notebook. Lab Archives is a multipurpose application that allows you to share your data with colleagues...other nice features. This app also has a great educational section. This section holds the Gibco Cell culture...directions including which buffers to use and that indicates if a sequential digest is needed. The enzyme finder... to this app; the user can search by sequence, category of enzyme, or name to discover all the information... Addgene.org Find Protoctols for Your Research Catch Up On Your Molecular Biology Basics ...

...together a grant application? Are you familiar with the publishing or patent application processes? How ... pages in a Curricula Vitae to showcase your publications, academic performances, and research experiences...information. Think "skills," not just "research and publications” What are the skills you developed through your...and writing was how I got my current job as a Communications Coordinator. Wondering how you can develop ...So You Think You have Skills.   Get Science Communication Practice with a Student Group Like Science in...Blogger! Theresa Liao (@theresaliao) is the Communications Coordinator for the Department of Physics &... a research career, she transitioned into a communications role instead after she completed her MSc in...

...ITRs serve as a type of origin of replication to initiate replication of the dsDNA virus. Viral packaging...plasmid while the other plasmids with packaging or replication components are called helper plasmids. Below,... production, they can be helpful for certain applications and aid in ease of use. Our lentiviral guide...AAV are rep and cap. Rep is required for viral replication and also aids in infrequent genome integration...a helper plasmid and mediate efficient viral replication. More information on these viral genes is available...particles. E2A/E2B are responsible for viral replication, with E2B being the primary viral polymerase....is missing the endogenous E1 gene, making it replication deficient. This is typically supplied by the ...

... its function, mimic a biologically relevant modification, or add in fluorescent or chemically reactive...function relationships or, in more practical applications, allow a protein to be more effectively used... you do to any number of experiments with. The catch The rapid expansion of GCE led to the creation of... of many new tools and potential applications. But this focus on development, along with the inherent ...’t give it a try. Optimization Why is GCE so complicated? Well, while the techniques are straightforward...ligation through introducing ncAAs with protein modifications of interest or any of a huge variety of ncAAs...has “10x the impact of PCR. But it’s 100x more complicated.” And Ryan Mehl would know. He’s the director...

...Our educational content  Have you ever wondered how we choose topics for the Addgene blog? Or why we...everything else we do! The goal of Addgene’s educational content Addgene truly believes that sharing speeds...antibody and content team, we identified what applications should be included and what basic information... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise...through an editing process and final check before publication.   Do research! Nearly everything we write entails...Guide figures. This requires a fair amount of communication between our content team and our graphic designer...graphic designer upon request that the science communicator "just sketch a western blot out quickly." Final...

...your application a whole week before the deadline. The day of submission, review your application with... their dream.  To help students focus on the application process, and simplify access to academic resources...Scrutinize every opportunity prior to starting an application to assess your eligibility, interest, and career... detailed. Beyond the resume, a cover letter communicates your interest in a specific program, and institution...February represent peak season for internship applications. Thus these noble individuals are inundated ... with a stellar letter of support.        5. Application submission  Set a calendar reminder for every...about outreach among low-income communities to educate parents about resources that can help their children...

...discuss automated protocols. 12:00 PM--Catch up. Finish any communications with my UK team members before they...getting to talk to so many different people! I communicate with customers, software developers, as well ...from interacting with others, and have strong communication skills to tailor your message to any type of... to solve is clear, but the solution is more complicated. You’ll need to be creative to be able to work...team in the UK is five hours ahead, so I’m always catching up in the morning! 9:00 AM--Technical implementation...out. 4:00 PM--Respond to customer help tickets. Catch up on customer help tickets, and respond when I ...technology, software, and science. She loves to advocate for the customer and help build software and technology...

... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing...Cas9 ortholog, and the target sequence must be located adjacent to that PAM. To scale up fluorescent CRISPR...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live... do not induce genome editing. If used with catalytically active Cas9, the short, CRISPRainbow gRNA will... PMCID: PMC3918502. Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor...PMCID: PMC4364232. Find the plasmids from this publication at Addgene. 3. Ma, Hanhui, et al. “Multiplexed...PMCID: PMC4864854. Find the plasmids from this publication at Addgene. 4. Kiani, Samira, et al. “Cas9 gRNA...

...from the serum. Protein A/G purification Protein A or Protein G purification is one common method. These...reagent that works for your species. Affinity purification Only up to 5% of the IgG antibodies produced...specific for the immunogen. A different type of purification can be performed to isolate antibodies only ...inexpensive. Their short production time and the bulk purification steps keep the cost low compared to other antibody...are particularly useful for certain research applications. Many secondary antibodies are polyclonal, as...target protein thereby greatly increasing signal amplification.  When to stay away from polyclonal antibodies...2018-0065 Fishman JB, Berg EA (2019) Antibody Purification and Storage. Cold Spring Harb Protoc 2019:pdb.top099101...

...thing that comes to mind. However, ion-based communication is not just for neurons. Non-neuronal cells ...It's just that for non-neural cells, ion-based communication happens at a slightly slower scale.  All living..., and when gap junction-mediated electrical communication is briefly inhibited in the entire worm during...reporting and genetically encoded fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric states...activated proton pump) and ChR2 (blue-light activated cation channel) light-activated channels to hyperpolarize...channelrhodopsin used as a sensitive fluorescent voltage indicator with near-complete desensitization in continuous...extremely exciting emerging field, with many implications for regenerative medicine, evolution of body...

... antibodies may be indicated on occasion, usually after other signal amplification techniques have been...IgG2 or IgG4. This can be important for some applications. Assay use While secondary antibodies are useful..., the flexibility of their use varies across applications. Western blots, for instance, almost always ...For the latter three, secondary antibodies are indicated when the protein of interest is found in low amounts...been ruled out.  Ease of access Signal amplification isn’t the only benefit of secondary antibodies. Finding...quantitative analysis of a sample. Instead, relative quantification can be done using either the direct or the ...antibodies remain the go-to option for signal amplification while providing needed flexibility in assay ...

... work around this is to "domesticate" your fragment: PCR-based amplification can be used to create silent...and exciting applications, this system is an incredibly powerful tool for cloning complicated constructs...Type IIS sites (such as BsaI or BbsI), that are located on the outside of the cleavage site. As a result...plasmid (Option 1) or added using PCR-based amplification (Option 2). Advantages of Golden Gate cloning...restriction sites that may not be amenable to "domestication", you might want to consider using an alternative...high-efficiency step. References: Sequential amplification of cloned DNA as tandem multimers using class-IIS...

...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming...particles and the virus particles produced could not replicate. However, Mother Nature found a way to re-create...packaging plasmid forming a vector that could create replication competent virus. Even though this system was ...still a potential risk for the generation of replication competent lentiviruses (RCL) especially if you...factors in HIV positive cells could make the virus replication competent. The 3rd generation of lentiviral...transgenes (>8 kb) Potential for generation of replication-competent lentivirus (RCL) Efficient gene transfer...transfer Potential for insertional mutagenesis: Replication-incompetent lentiviruses with human tropism can...

...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...expression profile at multiple steps from plasmid amplification in bacteria to transgene expression in vivo.... the level of spontaneous recombination. This indicates that the amount of leaky expression can largely...are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means that all DIO...of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of scrambled cre-recombinase...expression of receptors, post-translational modifications, and other factors that AAV utilizes to gain...

... a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...the sensors discussed below fall into this GECI category.   For calcium sensors, one size doesn...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophysical journal...neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor Protocols. 2012 Jun 1;2012...signaling using genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources...

...host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection... tools, thought was given on how to keep the replication of virus and its infectivity in check. Engineering...envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third plasmid... this safety feature prevents the virus from replicating and spreading in non-target cells, including ....    Viral cargo-specific safety   After replication competency, the second concern with virus safety...diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) and gamma...decontaminating liquid waste, remember that the percentage indicates the proportion of the decontaminate in the waste...

...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing...PMID: 26678082. Find the plasmids from this publication at Addgene. 2. Dalvai, Mathieu, et al. (2015...633. PMID: 26456817. Find plasmids from this publication at Addgene. 3. Zhang, Chi, Xiuhua Meng, Xiaolei...

...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...Select the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate... or introducing mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...Cas9-gRNA complex and delivered to cells using cationic lipids. Transient expression of CRISPR components...below: Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed...

...up. Applications of photoactivatable fluorescent proteins Some of the most important applications for ...conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible photoactivation...photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation Irreversible...level of background fluorescence. For any PA-FP application, the brighter the fluorescence the better. More...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...photostable green and red states. Beyond these imaging applications PA-FPs have been developed for use in yeast,..."Photoactivatable genetically encoded calcium indicators for targeted neuronal imaging." Nature methods...