...grant. Here’s a list of tools and resources that might help make writing a little easier. Writing schedule...when writing for a general audience. The tool highlights jargon terms or words rarely used in general ...frequency of use in orange, and common words with high frequency of use in black. This paper talks more...either based on the section of a manuscript they might be found, i.e. introduction, methods, results; or...Mendeley is free to use. You can take notes and highlight the papers saved to your Mendeley library. The...have a lot of the same features, but this chart highlights the differences between these two citation managers...

...the left while the SnapGene powered map is on the right (click here to see the new map in action). Read ...Analyze your plasmid sequence For more immediate, high-level analysis, you can click directly on the “Analyze...number of times they cut. Use the filters on the right to find single, dual, and 3+ cutters. You can also... on a particular restriction enzyme here also highlights it in the “Map” and “Sequence” tabs. Features...types. Clicking on a particular feature here also highlights that feature in the “Map” and “Sequence” tabs...direction. Clicking on a particular primer here also highlights it in the “Map” and “Sequence” tabs. BLAST...

...Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (>8kb) High Transduction... it encodes. This ability to infect cells with a high efficiency and deposit a DNA/RNA cargo into their...than the alternatives? Well, for one they have a high transduction efficiency and can effectively infect...organisms. Finally, viral delivery is typically straightforward across applications, while electroporation ...upcoming post. If a viral vector seems like the right approach for your experiment, you’ll need to decide...packaging is less efficient when inserts are on the higher end. Adeno and adeno-associated viruses Adenoviruses...do. They are generally used to achieve transient, high expression of the relevant cargo without the fear...

...and on-targeting likely more efficient.  These insights are hard to appreciate without computational support...must be confusing for a newcomer to decide on the right software to use. With so much choice the question...something akin to a frenzy as the crowd tries to weigh and determine the value of every sweet or toy strewn... audience blindly receiving them; it results in highly similar content and the absence of iterative design...result, the software piñata has been filled with slight variations on a theme without many resources to...tools The goal of this post is to provide some insight into available CRISPR software tools, what problems...various tools are trying to solve, and finally how we might proceed in the future. When we think of designing...

...two appear on an agarose gel at notably higher molecular weights than expected, given the size of the plasmid...plasmid can exist on as a dimer, depicted on the right, with the entire monomeric sequence duplicated in...), direct repeats (Ribeiro et al., 2009), and at high copy numbers (Williams, Carnes, and Hodgson, 2009... monomers, they are also able to replicate at a higher frequency (Summers & Sherratt, 1984; Summers et...are larger than monomers and will therefore run higher on an agarose gel (Bedbrook & Ausubel, 1976). In...2011) indicated that strain HB101 produced the highest yield of monomeric plasmid compared to DH5α. However...: 7029308. Goebel W, Helinski DR. Generation of higher multiple circular DNA forms in bacteria. Proc Natl...

... standard antibody: An ~150kD protein with both light and heavy chains made by immune systems. But here...imaging applications, smaller affinity reagents means higher resolution. Many of the below are also small enough...the lab, instead of generated in vivo, they have higher reproducibility and consistency in the final product...cleavage. They are comprised of the variable heavy and light chain regions, connected by a short linker peptide... longevity compared to antibodies, along with a higher likelihood of aggregation (Bates, 2019). They are..., are derived from a camelid antibody lacking a light chain, called a heavy chain antibody (Muyldermans.... DARPins typically consist of 3-4 repeats, have high specificity and binding affinity, can be produced...

...supervisor might expect weekly or biweekly progress reports. However, a hands-off busy supervisor might get ...Know-it all experts, who think their way is the only right way, and Indecisives, who keep changing their ... your discussion will get side-tracked, and you might create even more conflicts. 4) Define in advance...about mutually beneficial solutions Your supervisor might not agree with all of your ideas, but he or she ... conflict is miscommunication. For example, you might misunderstand your supervisor’s suggestions and ...sense their tone of voice in an email, there is a high likelihood of miscommunication, which can lead to...assertive to get your point across. Some supervisors might try to intimidate you with negative comments (hostile-aggressives...

...information from targeted amplicon sequencing is highly sensitive with detection levels as low as .01% ...of <4 bp. The off-target list is also generally weighted by the position of the mismatch along the length...detect double-stranded breaks and do not require high sequencing read counts making them fast and viable...Searches the genome for all DSBs, doesn't require high read counts, fast multiplexing Requires delivery...cells Not as senstive as other unbiased methods, high background BLESS Not reported Cell-based (in vitro...component required (e.g. dsODN), doesn't require high read counts (fast multiplexing) Requires large cell...can evaluate and validate off-target sites in a highly accurate manner. Using both of these approaches...

...technologies, one might incorrectly relegate RNAi to history. The straightforwardness of RNAi is unmatched—unlike...considered the “gold-standard” for LOF studies, the high gene copy number found in plants makes traditional... DNA, thereby limiting CRISPR’s effectiveness at high copy numbers and genomic loci with low accessibility...directly compared CRISPR to RNAi in the context of a high throughput survival screen found that either technology...for cell growth. Together, this excellent work highlights the importance of using orthogonal methods to...Arguably the greatest advantage of RNAi is the straightforward nature of the technique—all that is required... long hpRNA with Gateway-based cloning and straightforward positive selection of plants undergoing RNAi...

...provided some insight on these differences in viral production and will help you decide which is right for you...available, or the cost of custom viruses are too high for your budget. In those cases, you’ll need to ...more common to make lentiviruses and AAV in-house. High-titer adenoviruses are difficult and time consuming...be produced in a greater volume and will have a higher titer than a P1 virus. Pro-tip! Centrifuge the ...this is only possible because baculoviruses are slightly less prone to recombination than other viruses...Processing Processing of your baculovirus is straightforward — simply spin down the supernatant in a centrifuge...the next viral passage, titer the virus, or go straight into your viral application, like protein production...

...in a workable direction quickly. Of course, you might have less time to explore scientifically since you...industry, which means you’re more likely to have the right contacts and get your resume pulled to the top of... job applications require teaching experience or high numbers of publications, which may take a longer...chances of getting a good manager do tend to be higher in an industry setting. Scientific community and...and mentorship: As an industry postdoc, you might find yourself feeling scientifically isolated if no... have an in-depth discussion about your project might be challenging. In an academic environment, surrounded...re more likely to give an oral presentation to highlight any new data or ask for additional resources, ...

...protein Nucleic acids Nucleic acids Throughput High High High Efficiency Medium Medium Medium Difficulty...acids or protein Throughput High Very low Efficiency High Very high Difficulty Very easy Very hard...product in mind, steps 1 and 2 will generally be straightforward. Likewise for step 4, as this ties directly...a mammalian cell line that can be transfected at high efficiency. Lentiviral transduction Cas9 and gRNA...been published as CRISPR testing tools: Traffic Light Reporter System: Can evaluate lentiviral component...

..., we've looked back on the year would like to highlight some of the top articles from the Addgene blog...-CoV-2 protein expression, and more. We also highlighted reagent repositories and other collaborative ...time so that you can begin your cloning experiment right away. And without further ado, here are the top ...about hosting multiple plasmids in one cell, it might be that you can't stably maintain them in the cell...free molecular biology tools out there! Here, we highlight a subset of these tools for plasmid mapping, DNA...

...purposes Rights to all research results, modifications, and inventions created by the Recipient Right to ...terms, so you can rest easy knowing your rights – and the rights of your institution – are protected while...scientist working hard on research, and you need the right experimental materials. Who has time for paperwork...materials. Questions about intellectual property rights, pending publications, and commercial use often...patent applications on modifications or inventions Right to publish without editorial comment or review by...order, Addgene generates the MTA and emails it straight to you, or we can send it directly to your institution...

...marched in solidarity with our American neighbours, the fight is far from over here at home.  The work...of which impact reproductive rights and justice. Whether it’s used to fight the myths that abortions cause...wishful thinking. The March for Science is about fighting scientific partisanship, including more scientists...there's been a disturbing trend in the number of high profile people denying science, from celebrities...convince me to spend a Saturday in the tourist-trap nightmare that is Times Square. But I made the sacrifice...Most people won't argue with that idea, but they might feel disconnected from the entire scientific process...I think many of us grew to assume that the data might speak for itself but we're now in a rift where our...

...RNA molecules, which could bind their targets with high specificity and affinity, were eventually called...nucleotide-based ligand binders would prove to be highly adaptable tools. The key to aptamers’ flexibility...conformational changes after target binding, aptamers are highly customizable in terms of how they actually “report...applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing with veggies) Though...eventually evolved into Spinach2, a more stable and brighter version of the aptamer, but it still had limitations...more stable in cells. The new aptamer also has a higher melting temperature than its Spinach ancestors,...have also made aptamers an important component of high throughput assays with microarrays and biochips....

... client situations might be especially challenging. For instance, the client might need to get a large...what a career in this field involves, and why you might enjoy doing it. I’ll also talk about the skills ...and anticipated commercial success. Or the client might need help with the intricate task of designing a...topics are typically cutting edge; a consultant might outline the impacts of combining targeted molecular...turnover of projects, pressure to maintain consistently high levels of performance, and occasionally being faced...cases to better staff future teams. As with any highly collaborative work place, challenges can arise ...or medical device works. This understanding sits right at the core of the suite of benefits a given therapeutic...

... the high school interns as equals, because as soon as I had passed my training, I dove straight into ... never would have imagined that my summer before high school senior year would have been spent within ...weeks that we had made a mistake by not having the right concentration of a reagent during our HiFi assembly...the following two weeks, we were able to obtain eight clones. We ran digested and undigested samples of...one clone we found with the correct bands was the right plasmid, so after discussion, we sent it for sequencing...invited guests came to see the research from all the high school interns, done within the past six weeks. ...other way. Toj Toj-Villagran is a rising senior in high school who just completed their summer internship...

...activation (chemogenetics) or light (optogenetics).   Timing Optogenetics offers a high degree of temporal resolution...presence or absence of the correct wavelength of light. This response is immediate, on the scale of milliseconds...tissue-subregion targeting down to the limits of light delivery. Chemogenetic approaches excel at broad...offers better control over these factors as the light source can be easily and quickly manipulated or ...Addgene website. Optogenetics also requires that light of the correct wavelength reach receptors in the... address this is by working with organisms with light accessible brains, such as zebrafish larvae. Mouse...implant to be surgically installed to allow for light delivery. Chemogenetics poses the advantage of being...

... tips for high efficiency cloning with this technique: Amplification efficiency is highest when using...labs, a variety of other cloning techniques with higher efficiency and simpler procedures have been developed... in the figure from Chaokun et al., 2011 to the right. After PCR, you should confirm that the products...introduced during PCR or any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations...chance of introducing an unwanted mutation. Tips for high efficiency cloning Now that you have a better understanding...using high fidelity DNA polymerases like phusion. An 18-cycle PCR leads to the most colonies after transformation...27391134. 2. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-and ligation-independent...

...fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for SLs in vitro...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...organoids. The Organoid EasyTag System is a straightforward workflow for doing gene targeting, reporter.... This novel type of biosensors could be used in high throughput screening for agrochemical compounds ...CRISPR Plasmids and Resources page. Here are some highlights from the past couple months: A new lentiviral...

...neurons in the mouse brain. MouseLight images can be found on the MouseLight website. Together with scientists...neuroanatomy and neural connectivity, providing valuable insights for neuronal communication. Viral vectors and...vectors can be equipped with strong promoters for high and neuron-specific transgene expression. Fluorescent...for their production and purification that yield high titers (see also AAV protocols for production, purification...researchers from the Janelia Campus for their MouseLight project but neurons were traced with AAV2. Two-Photon...variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV serotype (Tervo et al....also having the added advantage of lower toxicity, higher specificity and reduced technical variability compared...

...forward, in this post I’ll point out some of the highlights and takeaways I got from the symposium. The ...outcome is not easily achieved. Of note, it was highlighted during the conference that just increasing funding...projects sought to make an interactive game that highlights just this point. As part of this, trainees need...thoughts The FOR Symposium Boston 2015 shined a light on some of the many problems facing the academic...academic research enterprise. In addition to highlighting some of the important questions that still need to...

...with different properties (for example, overall brightness or on/off kinetics) that makes them suitable ...(pGP-AAV-syn-FLEX-jGCaMP7b-WPRE) exhibits the brightest resting fluorescence of the GCaMP7 family of calcium...pAAV.hSyn-FLEX.iGABASnFR) has good membrane localization and brightness. The F102G variant (pAAV.hSyn-FLEX.iGABASnFR....through imaging. Since it’s illuminated by red/orange light, Archon1 can also be used in conjunction with optogenetic... activators and inhibitors that respond to blue light. We recently released a constitutive (pAAV-Syn-Archon1...-jGCaMP7f-WPRE AAV1 with a CAG promoter exhibits high specificity for Purkinje neurons. 105321  AAV1...pGP-AAV-syn-jGCaMP7c variant 1513-WPRE 7c exhibits high contrast between peak fluorescence and resting fluorescence...

...Some common ones you might see include ColE1, pMB1 (which comes in a few slightly different but well known...make additional copies. ori sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs...make a lot of your plasmid? Is the gene toxic in high amounts? It is always good to keep in mind that ...propagate plasmids, but endA- E. coli are best for high yields of plasmids. Growth conditions The amount...culture inoculum Freshly streaked bacteria have higher copy numbers – for optimal results always pick ...cultures. Freshly transformed cells also give a higher plasmid yield that colonies streaked from a glycerol...stock. Incubation for 12-16 hours tends to give higher copy numbers since the bacteria have just reached...

...fluorescence changes or a requirement for very high excitation light intensities have made these indicators difficult...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...measure ApoB-LPs directly and develop a potentially high throughput in vivo model to identify drug candidates...Thierer et al, 2019). Zebrafish are a great model for high throughput study of ApoB-LPs because their ApoB ...amenable to use with other bEBP/UAS pairs, and displays high dynamic range. However, the promoter in question...lipid compositions of C. elegans membranes, which might beneficially influence the rhodopsin properties....

...projects in many labs. However, the high costs of sample preparation for high-throughput next generation (NGS...Addgene to develop a highly optimized bioinformatics pipeline that will return high-quality assembled and...quantities of library molecules for each sample remain highly normalized across a wide range of input DNA concentrations... vary by DNA input amount. plexWell™ + Addgene = high quality NGS-assembled plasmids Because of the enhanced... plasmids. The plexWell™ workflow also offers a highly efficient NGS-based alternative to Sanger sequencing...assembly pipeline. To date, this has resulted in high quality NGS-based assemblies for over 3000 plasmids...

...While I had always enjoyed learning about biology in high school, it wasn’t until I started working in my ...molecular biology-based activities making their way into high school classrooms, like gel electrophoresis to separate...led me to develop BioBits ExplorerTM and BioBits BrightTM, biology kits based on freeze-dried, cell-free... that students can easily sense, whether that’s bright fluorescent proteins that they can see, enzymes...initially developed the BioBits kit to be used in high school classrooms, but it could be widely adapted... a biology teacher at the elementary, middle, or high school level, feel free to check out one of our ...PMC6070312. 2. Stark, Jessica C., et al. "BioBits™ Bright: A fluorescent synthetic biology education kit....

...Antibodies 101: Choosing the Right Isotype Antibodies 101: Selecting the Right Antibody Resources on Addgene.org...groups on a western blot and look for the molecular weight shifts indicative of tau phosphorylation. By including...protein standard (P) to check for tau molecular weight shifts. A negative control is a sample or ...runs. If the results vary considerably, then the high degree of variability in the process could make ...signal from your target needs to be significantly higher than the background noise (Waters 2009). If the...the fluorescence from your target is extremely bright, then the noise likely will not interfere. If the...control for all new samples, protocol changes, or when high background labeling occurs (Burry 2011). Complex...

...commonly used, PVDF tends to be slightly more popular due to its durability and high binding capacity. In addition... (HRP), that emits light upon reacting with a specific substrate. The emitted light, or chemiluminescence... samples, a protein standard of known molecular weight in a separate lane can help confirm the size of...acrylamide content of the gel. For small proteins, use a higher percentage of acrylamide to increase migration ...broad range of protein sizes but takes hours to overnight.  In contrast, a semi-dry transfer only requires...chain. Consequently, antibodies validated for IP might not work for a western and vice versa.  In addition...specific experimental condition you could look for a slight size shift in the protein band before and after...

...junior faculty member at Harvard Medical School/Brigham and Women’s Hospital (BWH). Based on her experience...years ago looking for their career improvement) and bright students that will be the future of the country...to help build, together with the universities, a bright future. IMP provides matched one-on-one international...Although all mentorship relationships are valuable and highly recommended, a mentor with international experience...impressions of the IMP experience. Conference highlights included remarks by the Spanish Secretary of ...representatives from different Spanish universities and high level public and private institutions also showed...website Learn about the Mentor Circle Program at Brigham and Women's Hospital Visit the International Mentoring...

... be on LinkedIn?” For anyone who is planning or might need to plan for a career outside academia (that...business or follow up. Use the "Contact info" link right below their photo. 2) Personalize all connection...note, you waste an opportunity to start things out right. Also, many people on LinkedIn don’t appreciate ...connect button unless you’re on a profile page. You might be looking at a list of suggested connections and...connect people that want to meet or that you think might be helpful to each other. This is a quick way to... before you’ve had a chance to proof read. This might not be important for a good friend, but if you are...and it’s still connecting with a stranger so is highly discouraged. You should be using your network to...

...applications and gene therapy: they can be produced at high titers, can accommodate large transgenes, transduce...Gutless Ad vectors (helper-dependent (HD-Ad), or high-capacity (HC-Ad__ are devoid of viral genes and ...belongs to this category. 3rd Gen AdV (Gutless or high capacity AdV) are devoid of all viral coding sequences...proteins in trans. The Gutless AdV have generated high interest for gene therapy due their increased cloning...more HEK293 cells and produce more rAdV to reach higher titers. Successfully infected cells will become...separate rAdV from other cellular debris. Purified high-titer rAdV is then dialyzed and stored at -80C. ...account. You're now ready to use your purified, high-titer rAdV in your experiment. Enjoy! References...

...midst of a crisis. The “antibody crisis” came to light as antibodies became commercialized, revealing the...goldilocks antibodies. A goldilocks antibody is highly specific (minimal binding to off-target molecules...creating a public and accessible database for low-cost, high-quality antibodies for IHC. Working with collaborators...their recombinant forms (rAbs). mAbs and rAbs have higher homogeneity than polyclonal antibodies because ...from the negative control, such as showing a very bright signal only in the hippocampus compared to other...the amount of time the sample needs to be under light to discern the signal. If the exposure time is long...allowing scientists to find and utilize reliable, high-quality antibodies. We are creating a database for...

...catalog to help you find the right split-FP plasmid for your experiment.  Highlighted Split-Fluorescent Protein...bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Kodama Y, Hu CD. Biotechniques...amino-terminal acetylation system and novel in vivo high-throughput screen for molecules affecting alpha-...Improved sfCherry3C(1-10) for use with sfCherry2(11) Bright split red fluorescent proteins for the visualization... chromophore formation (abundant in eukaryotes) Bright monomeric near-infrared fluorescent proteins as... with exogenous fluorophores, which can even be bright enough for single-molecule tracking in live cells...

...PAM) might work too.  High dCas9 and gRNA expression are key for complete DNA demethylation – tight binding...the Future, and big hair. To what was perhaps a slightly smaller population, something else was also suddenly...conference or a karaoke bar), you probably want to sigh. The problem is that many of these studies report... 2018). This means that off-target loci – in neighboring chromatin or throughout the genome – could be...reality, editing your target site is not as straightforward as expected. TET1 can have many effects independent...and pLM-CMV-R-Cre. Selection for cells stably and highly expressing these constructs can be achieved by ...

...injection site is straightforward, as it targets large muscle groups (usually the thigh), though it is important...injection, like you might after getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27...use depends on a number of factors. You have to weigh the pros and cons of various methods and choose ...circulation is slower, and more institutional oversight is required. Restraint: mechanical restraint or...such as experimental therapies or pain relief, straight to the stomach. This technique involves administering...Intermediate Intramuscular Mechanical restrainer Thigh 25–27 < 0.02–0.05 mL Beginner Subcutaneous Back...

...emission spectra, meaning they will emit fluorescent light at similar wavelengths (See this handy emission ...against APC-Cy7, the populations are in more of a straight line and are only positive for APC (Figure 1B)...horizontal line, so our circled population on the right needs to shift towards the X axis to become a perpendicular... plot in 2A, the populations are not perfectly straight, but the center of each population is in a line... sample you are trying to compensate in the top right corner of a flow plot, meaning that it should be..., it will show up as a diagonal line in the top right corner of the plot and large compensation values...positive sample. The cell population in the top right corner of the plot is positive for both markers,...

...for fusion proteins! Despite multiple studies in high profile journal articles, many researchers remain...primarily as a dimer in cells while a protein with a high micromolar or low millimolar Kd is unlikely to form... Following the simple logic outlined above, you might think EGFP is unlikely to form dimers in a cell....then their effective concentration will be much higher than if they are homogenously distributed and mobile...concentration of FPs around that fusion protein is very high. Real world examples of these situations in cells... the POI. These structures are large (microns), bright, non-physiological, and difficult to ignore [1,...normally self-associate into homodimers or even higher order oligomers. Unfortunately, FP dimerization...

...and understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification...categories, arriving at a consensus was not as straightforward as it seems. There is not a single gene that... sequence similarity, phylogenetic analysis, neighborhood analysis of surrounding genes, and experimental...cutting up DNA after recruitment by Cascade. As you might imagine, this is handy for creating large genomic...adaptations for increased fidelity, activation/repression, high precision edits, and more. Figure 3: Schematic...Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting edits using...with BioRender.com.   What’s your type? It is highly unlikely that you will have to know and understand...

... completed with the Ig light chains. The variable region of the heavy and light chain determine what antigen...going to spot the whole, long word right off the bat, but you might see a few letters and be able to guess...Two immunoglobulin (Ig) heavy chains and two Ig light chains make up each antibody molecule. The Fc region... of an antibody including Fc, Fab, heavy chain, light chain, constant region, variable region   The...made up of the variable regions of the heavy and light chains fused together to form a single protein that.... The proteins are separated based on molecular weight by SDS-PAGE, and are subsequently transferred to...interest and detect its presence. Then, the amount of light that can pass through each well - the optical density...

...accurate and reproducible. I also highly recommend spending the money on a high quality stereotaxic frame with...and versatility, the technique is remarkably straightforward to learn and relatively cheap (compared to ...manipulated a number of the opsin genes to alter the light spectrum for activation and the kinetics of opsin...above) include Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation...potential. Halorhodopsins (HR) are green/yellow-light sensitive chloride ion pumps, activation of which...expression alone is extremely limited. Therefore, highly expressing, generic neuronal promoters such as ...privately communicated to me as one of these extremely high expressing promoters). Conduct a time course experiment...

...and writing are key skills. Other fields, often highly technical or skills-based ones, may not use them...are less likely to require cover letters, while higher-level jobs are more likely to consider cover letters...that asks for strong organizational skills. You might think, “I’d be good at that! I organized my lab ...]. Notice that the intros above allow you to highlight what specific skills, qualities, or interests ...considered cover letters to be more important for higher-level positions. Many did not require cover letters...-neutral greeting like “Dear Hiring Manager.” Highlight your achievements with specific examples. Instead...

...parts of our code, we might be calling something a sample and in other places, we might be calling it an aliquot...soundness of the software design. Each of us brings slightly different cognitive biases as a result of our ...creates trust amongst the team. Whether we are a highly collaborative team because we are using the Scrum...we are using the Scrum framework because we are highly collaborative, we love teamwork. Working collaboratively...parts of the shipping code. The following sprint we might have an EMTA theme and we will all be working in... Most of the time, updating these libraries is straight forward. However, there are times where a library...no further updates, security or otherwise. This might sound like Greek to you, which brings me to my next...

...densities that are low enough to prevent confluency but high enough to promote cell growth. Overgrown cultures... age of your culture post thaw. In general, the higher the passage number of a culture the lower the transfection...be a bit dubious due to lack of documentation and high turn-over of lab personnel. If you are unsure of...with pH changes and resource consumption due to the high cell density of the culture. In addition to benefitting...harvest approach is that it tends to result in a higher volume of lower titer virus. In such cases you ...downstream applications. While this ensures that the highest titer virus is used, it is not always feasible,...titered. Most titering approaches transduce into highly permissive cell lines such as HEK293, H0 or A549...

...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today in our transferable skills...with SnapGene, for example, allows us to display high-quality maps and annotations without us having to...events can be very tough. First I needed to find the right contacts at the companies and then convince the ...Great progress! You are collaborating with your neighboring lab, you've met interesting people at conferences...are the individuals that are part of them. I’m delighted that these collaborations give me opportunities...An Imperative for Graduate Students" from Inside Higher Ed Additional Resources on the Addgene Blog Check...

...have limited use in vivo because they are based on light which is very easily scattered by tissue. Ultrasound...modality with good tissue penetration depth as well as high spatial and temporal resolution, making it ideal...vesicle genes from Serratia sp. 39006 produced the highest levels of nonlinear acoustic contrast in E. coli...). Gas vesicle expression in vivo is also straightforward: after the E. coli have colonized tumors, inject...Methods to assess bARGSer expression, from left to right: colony whiteness on inducer plates, white inclusions...We wanted to create second-generation mARGs with higher expression, a smaller genetic footprint, and nonlinear...chromatin-opening agents. It ended up being very straightforward to engineer various cell lines from different...

...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...bleaching to allow for high-resolution imaging without degrading the tissue (Radtke et al., 2020, 2022). I do... do mean highly multiplexed: you can image over 65 different parameters using this method (see Movie S8..., you’re probably also very interested in having high-quality antibodies and reagents that you know work...you’re certainly not alone – we’re all looking for high-quality antibodies and reagents that work for our...immunolabeling and chemical bleaching method for high-content imaging of diverse tissues. Nature Protocols... Data with Addgene Antibodies 101: Choosing the Right Isotype...

... for a reply to an email. You need answers: the right ones, quickly, and this is why we have a dedicated...phone call or write out an email. You need help right at the moment you’re looking for it, or you may ... from your colleagues, to intellectual property rights and handling your constructs once they reach you... new feature on our website; towards the bottom right corner of each page there’s a blue icon displaying...result. By clicking on the result links themselves right from within the widget, you’ll navigate to articles...on the "Leave us a message" button shown to the right, you will be be prompted to enter your name and ...team’s attention, where we can then offer the same high level of direct service you’ve come to expect from...

...together DNA fragments. As a fun way to start, I highly recommend watching this entertaining video created...assembly work? The required homology between neighboring fragments can be created via PCR amplification...including regions of homology to the vector or neighboring fragment. Then you would amplify the fragments...success rate of this reaction is typically fairly high, so there is usually no need to screen a large amount...structure, such as a hairpin or a stem loop (as might be anticipated to occur within a terminator sequence...required single-stranded annealing and priming of neighboring assembly fragments. Gibson assembly is commonly...vector that you want to use is very large, has a high GC content, contains a lot of repeats--any of which...