...upper right) and a mixed population of B cells and dendritic cells (DCs) in the lower middle right. DCs...generated often looks like the graph on the left, it might make more sense once you realize events are plotted...plotted on an invisible Cartesian plane (right.) Each event is plotted by the amount of signal read from...cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive for...another two are B+. Our A-B+ cells are in the lower right corner.      Figure 2: This flow plot has... (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell, instead of fluorescent...DCs), mDCs and pDCs. Let's follow it from left to right.  First, SSC and FSC are measured to differentiate...

...FRET signal you should choose the highest quantum yield donor, the highest absorbing acceptor and fluorophores... similar brightness - even though in the most popular pair, CFP has five-fold less brightness than YFP...that case the cross-talk is high and your background signal may be higher than the signal generated by... the acceptor (linking the quantity of absorbed light, at a given wavelength, to the concentration of ...that can affect FRET measurements include: the brightness of a fluorophore pair, donor:acceptor stoichiometry...the donor is excited by a specific wavelength of light and the signal is collected by using emission filters... of the donor is increased. This method is straightforward and quantitative, but it is destructive and...

...insertional effects in the genome. (Bottom panel, right) A highly expressing RUBY plant is a vibrant red/magenta...transparent tissues, or in green tissues is slightly darker or slightly brown. Photos by Andrew Willoughby ...something new has been added to the palette. RUBY is a bright red pigment reporter whose expresion is visible...RUBY as a selection marker The creators of RUBY highlight its use in crop plants under field conditions ... further experiments.     Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following floral...from the same plant beginning to germinate. (Top right, second panel) Transformed seedlings can be selected...

...and it emits light that peaks at 508 nm. The protonated state, however, does not absorb light at 488 nm.... efforts have increased the brightness of these genetically encoded light emitters (i.e. Takai, 2015 and... enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another mutation (A206K) is necessary...of the available green fluorescent proteins emit light. The pKa of fluorescent proteins can be accurately...that are engineered to bind specifically and with high affinity to fluorophores. Examples are the FlAsH...fluorescence overlaps with autofluorescence The cyan light that is used to excite GFP, may also excite several...shifted to longer wavelengths. Therefore, the use of bright red fluorescent proteins (e.g. mScarlet) or infrared...

...illuminates the passing cell with a laser and detects the light scattered by that cell at a low angle (FSC) or large...voltages to observe your cells on a flow plot, you might have to do some testing or ask experienced colleagues..., morphology, and structural complexity.   Height, Width, and Area Within FSC and SSC, there are ...are three parameters that are being measured: height (H), area (A), and width (W). The different measures...few events at the end of your gating pipeline, it might be worth going back and loosening the FSC/SSC gate...Figure 2), single cell events will show up in a straight line one the plot, while duplicates deviate from..., it can depend on the type of stain whether a tighter or looser gate makes sense. Here, luckily, the ...

...listen, and an ability to remember the right stories at the right time. Like many of the posts in our ...be considered in the right, and one in the wrong. If you arbitrate, one party might get exactly what they...I first realized I might have some minor skills in conflict resolution after college, when I discovered...inclination for this skill that you could strengthen. The right set of transferable skills, coupled with your technical...primary role is customer support, but there are eight other teams who still retain a customer support ...be wary of anyone who is actually looking for a fight. But you also can’t be the type of person who avoids...or invalidated. Both compromise and arbitration might remedy short-term substantive conflicts, but they...

...total protein are compared. While a higher slope indicates higher sensitivity, linearity is the most important... quantification of a western blot — and why you might want to be careful relying on so called “housekeeping... simply reports which POI band is stronger. The right gel has exactly the same POI bands as the left gel...plot of protein abundance versus band intensity (right). Created with BioRender.com. On this gel, the... the protein of interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the...under your experimental conditions. Otherwise, you might want to consider a different loading control. Assumption...accurately quantified Most housekeeping proteins are highly expressed in cells. This means that at sample concentrations...

...the PCR process, you need a high-fidelity polymerase. There are many high fidelty polymerases on the market...Cornell University. Site directed mutagenesis is a highly versatile technique that can be used to introduce...SNPs in a plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis...site-directed genome editing requires a template of high homology to the endogenous target, yet to facilitate...Restriction Fragment Length Polymorphism (RFLP) analysis (right). Introduction of restriction site “A*” results ...rather than a linear product (see figure). Template A high purity plasmid prep significantly increases the ... target site, can identify these duplications (slightly larger bands sizes relative to the original template...

...complete desensitization when exposed to continuous bright light. Desensitization is mediated by a conserved ...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...MerMAIDs, neurons can be turned off quickly and with high precision - a valuable optogenetics feature when...PubMed PMID: 31346176. Using LADL to loop with light Article contributed by Eric Perkins Mammalian genomic...looping, the Phillips-Cremins lab developed the light-activated-dynamic-looping (LADL) system. The group...protein that heterodimerizes with CIBN upon blue light exposure (see Fig 1*). When the group targeted the...increased Zfp462 expression within 4 hours of blue light induction (6-fold faster than previously developed...

...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...

...provides an example calculation of the weights. Table 1: Journal Weighting Example Article Number (A) Year... with Addgene Plasmids in Journal (F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature...associated with Addgene plasmid deposits are more highly cited, we went to one of the largest bibliographic...get our journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene ...they comprised 10 percent. Based on this new re-weighting, we can compare citations where an equal share..., you can see the full impact from using these weighted numbers. Note: this does drop some journals from...all other explanations, we do think that it lends weight to the argument that Addgene is achieving its goal...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

... 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...

...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...