...that onto a mass spec. That's very time intensive and expensive. There's the promise that we could instead...DNA systems capable of storing data and computing inside living cells. He's also a co-founder of the BioBricks...describing the development of “transcriptors,” transistor-like digital genetic switches that enable cellular...proteins. That's central to everything that goes on inside a cell. Until we get a handle on precise, forward...

...protein combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal...(Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and nMagHigh1...able to reach deeper tissues. Additionally, high intensity exposure can cause DNA damage. Far-red light, ...Michael Lin, who developed a chimeric protein consisting of Cas9 and a photo-dissociable dimeric green... anti-CRISPR Acr protein, AcrIIA4, to a photo-sensitive LOV2 domain from A. sativa (Bubeck et al., 2018...editing For more details, check out this comprehensive review comparing optogenetic CRISPR techniques...

...use a standard curve and provide some general considerations for scientists planning to use them in their...to 1:32. Each individual step of the series is consistent at 1:2. When preparing a dilution series, use...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...not fall within this range, it may be time to reconsider your graphing program. Summary Few things in ...run.  When setting up your dilution series, use consistent dilution factors, change pipette tips regularly...

...immune system directly). It may seem odder when you consider that out of the 6 x 106 virus-like particles that...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient... natural virus, this new chromosomal locus is considered “proviral DNA” (Rowe et al., 1971) and the full...

...of current new and exciting literature. As I transitioned out of academia and away from the bench I certainly... figure organization with a different person responsible for each figure. Others still are more casual...everyone to offer thoughts you may not have even considered. These questions should be tailored to the group... not actively participating, it may be time to consider ending the meeting. This is a common failing in...participants. If you find yourself in this situation, considering stopping (temporarily or permanently) the recurring...

...are some potential complications that must be considered when using this method:     Cells must be amenable...to directly deliver a ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting...gRNA from a commercial vendor, this can often be expensive. An alternative approach is to express and purify...is most appropriate for a given experiment. Considerations for RNP-mediated CRISPR experiments While using...with these types of experiments. One important consideration is that the Cas9-gRNA RNP will be degraded by...

... venture presents itself even outside of work, consider challenging yourself, take the opportunity, and... all the things for which you are officially responsible on paper, and take some time to write down all... have to exercise in order to manage these responsibilities. If you are in charge of completing many ...management and organizational skills. If you are responsible for working with lots of students, managing inventories...the short list of transferrable skills here and consider which of these skills you’ve used in your own ...

...immunoprecipitation (locus-specific ChIP). These methods consist of insertional chromatin immunoprecipitation (iChIP...Advisory Board.  Addgene: Your lab has worked extensively with enChIP systems. Can you describe this technology...technologies. The locus-specific ChIP technologies consisting of iChIP and enChIP are recent examples. We also...community is a kind of duty for researchers when considering the peer-review system of journals and grant ...Identification of Proteins Associated with an IFNgamma-Responsive Promoter by a Retroviral Expression System for...

...and it will teach them to respect their own responsibilities. Another tip: don’t miss your grandfather’... the evening, or in a few years) when life responsibilities are less You can work at home sometimes, but... the burden alone You, alone, are not solely responsible for care and attention to your home and family...doctor without your help. Your partner is equally responsible and capable. The only exception is that men cannot...tasks – try to resist the impulse to control by insisting things always be done your way. Be patient with...

... package stays in transit for a certain number of days and tracking average transit time for each courier...having some materials available to order, yet not consistently accessible, wasn’t an option. So we identified...For example, when we began offering temperature-sensitive materials, we noticed a large percentage of these...distributor allowed us to ensure that temperature-sensitive materials like viral preps and libraries were ...

...emerged from intensive engagement with a wide range of people at the University of Wisconsin in 2011/12 ...reproducibility is limited in two ways – only data inside the ELN is reproducible, and only the highly restricted...principles emerging from the engagement with the Wisconsin research community were to: (1) prevent vendor... choosing an ELN for your research, be sure to consider how its features do and don’t support reproducibility...

...resistant to antibacterial drugs. Resistance can be intrinsic, meaning a basic characteristic that naturally...resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma species’ resistance...possess cell walls. As a result, Mycoplasma are intrinsically resistant to this class of antibiotic. Acquired...be unharmed by the compound it produces (think intrinsic resistance), but surrounding microbes would be...prescribed to them This may seem counter-intuitive considering the other three scenarios, but a short treatment...

...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...activators The simplest of the dCas9 activators consists of dCas9 fused to a single transcriptional activation...Mediator complex, generally the strongest activator, consists of a dCas9-VP64 fusion and a modified gRNA that...

...AAV delivered genes. AAV serotypes have been extensively studied in order to define those that target ...weakly expressed. As these epitopes tags have no intrinsic visual signal, they cannot be detected directly...neurons. As compared to epitopes tags, FPs are intrinsically fluorescent and can therefore be used in live...darkened chromophores. These probes are both intrinsically fluorescent and can be detected with highly ...tags like GFP. Specifically for STORM, the high intensity signal provided by smFPs can result in better ...

...viral vector solution. Since this technique is intensive and requires an electron microscope, it is not...image shows that the vast majority of the vectors consist of full particles (white arrowhead) relative to...from different sources in the same experiment, consider retitering both AAV in your lab. The absolute ...does not give an indication of infectious titer, consider titrating each lot of AAV in vivo to determine...Additional Resources on the Addgene Blog Important Considerations When Using AAVs Addgene's AAV Quality Control...

...sure your cells have first reached an appropriate density. Cells in the exponential growth phase are alive... prevent the culture from reaching the desired density within a reasonable time-frame. For these cases... growth media, or if your plasmid is low-copy, consider reducing the antibiotic concentration. Growth ...accomplished by growing your culture to the desired density at 37°C and reducing the temperature or moving ...TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

...antibodies, increasing the fluorescence signal intensity. However, you need to be careful when selecting...interest is not located on the cell membrane but inside the cell. Due to their chemical nature, antibodies...nucleus intact. If markers of interest are located inside the nucleus, a different fix/perm kit is required...that can enter cells freely are typically used. Inside the cell, the ester group is cleaved off by enzymes... yielding a stepwise decrease in fluorescence intensity in a (histogram) flow plot (Figure 4b). Through...

...concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) ...vector with Gibson assembly. While this is a comprehensive mutagenesis method, a drawback to using Gibson...scanning libraries. PFunkel: Of prior existing comprehensive mutagenesis methods, PFunkel offers the best...PCR-based approach for generating a customizable comprehensive mutagenesis library that’s ready to be tested...Elad, and Marc Ostermeier. “PFunkel: Efficient, Expansive, User-Defined Mutagenesis.” Ed. D. Dafydd Jones...

...metabolic engineering.  The E. coli genome has an extensive research history and characterization. Many proteins...cardiovascular disease, specifically stroke and hypertension (Iannaccone et al., 2009). Rodents have also...amphibians in science with early research providing insight into some of the most significant and fundamental... plant model organisms in the world. With its extensive scientific history, highly characterized genome...Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493–521 ...

...gene of interest) and prevent incorporation of extensive sequences used for cloning into the final translated...Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium... integrity, expression level, and aggregation propensity in four expression systems: E. coli, wheat germ...frequencies across timepoints or populations. This kit consists of a 48 plasmid adapter library, with each plasmid...Morgan’s blog post on the PUF Assembly Kit for some insights on its potential from its creators. We hope you...