...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...

...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...

... 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 ...

...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...

...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...

...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...

...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 ...

...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...

...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...

...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...

...included inducible Cre, such as the tamoxifen-responsive CreERT, the coupling of Cre-lox with FLP-FRT ...limited due to low resolution or were very time-intensive, necessitating the development of a new technique...This system differs from Brainbow-1.0 in that transient Cre recombination is required to prevent constant...tissue was challenging due to low fluorescence intensity, caused partially by fluorophore photoinstability... the Brainbow technique will provide important insights into the complicated physical organization of ...

...prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid: the Escherichia...certain restriction enzymes that are methylation sensitive. dnaJ Mutation in a chaperonin gene Increases...strains are all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain...Dam or Dcm methylated, allows for methylation sensitive restriction enzymes to cut the plasmid after preparation...tables are by no means exhaustive! For a more comprehensive list and additional information, visit OpenWetWare's...

...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should..., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, although these...The off-target activity of gRNAs is important to consider. While the basic landscape of mismatches that ...

...FLEx Vectors Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...-target expression.  Some classes of neurons transiently express TH during development or have low levels...titer to prevent off-target expression of the sensitive FLEx-split TVA receptor used for tracing neural...recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors. Proc Natl Acad Sci USA 116:27001...

...the Moloney Murine leukemia virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing...vectors Although retroviruses are still in use, considerable efforts have been devoted to develop efficient...generation. The 1st generation of lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector... from one batch to another. For experimental consistency it’s a good idea to purify and titer your virus...sucrose to the supernatant to create a gradient of density that favours virus precipitation. My tip for this...

...for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...cooperate to cause a higher-energy electronic transition in a fluorescent molecule (see figure to the ...technical specificities have to be taken into consideration especially in the use of fluorescent proteins...wavelength can significantly affect emission intensities from fluorescent proteins (Salomonnson et al....Marcello, et al. "Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial...

...before you even receive the product? How can we consider the environmental impact of those processes? Who...laboratory products that emphasizes Accountability, Consistency, and Transparency. This label scores products...might sound risky in terms of contamination, but consider flow cytometers. These fluidic machines handle...through an EPA-approved process and no longer considered hazardous. Then, they use this material to produce...

...goes beyond such restrictions, you may want to consider a systemic capsid for your AAV.   Systemic capsids... definition of systemic capsids. After some consideration, we offer this definition: A systemic capsid...capsids are engineered, as well as highlight some considerations for their use. Then, we’ll provide information...and/or its intermediate engineered precursor. Considering systemic capsids for your research? While systemic...payload — there are a few additional, specific considerations you’ll need to be aware of before starting ...choice on which systemic capsid to use. Factors to consider include the delivery method, mouse strain, target...recommend you check out Caltech’s CLOVER Center’s extensive repository of published systemic AAV experiments...