...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...

...time consuming and expensive, especially when done in vivo. There are also sensitivity issues when attempting...them remain uncharacterized. This represents a considerable barrier to furthering research and developing...Atlas (TCGA), a catalog of genetic mutations responsible for cancer from genomics data, and large-scale...

...  Joining the Addgene crew!  This pattern was consistent: everyone I spoke with at Addgene, no matter ...been done at Addgene before. This meant I was responsible for establishing a new report and routine across..., and make the occasional TikTok. I was also responsible for typical entry level tasks such as stuffing...connections with coworkers seem, in retrospect, nonsensical. I have found the people at Addgene have become...

...need to be inside of a gene’s open reading frame, in fact, they can be activated a considerable distance... These reporters have historically been used extensively in fly, mouse, and yeast models. Why traps are... endogenous gene expression are all factors to consider. Once you’ve decided on the type of trap, there...

...interests. We've outlined some important factors to consider and summarize helpful advice from the successful...Achieve Success in Academia". Important factors to consider when choosing a research project After joining... your professional brand. It offers colleagues insight into your interests and will determine how you ...fungal biology.” Connie Cepko advises students to consider their goal of PhD training when selecting a research...

...offer more information on these regions.   Cas9’s transition from weak, random binding to precision gene editing.... This cap is where the DNA:RNA heteroduplex transitions back to DNA duplex, and it’s the reason guide...RuvC loop caps the heteroduplex and marks the transition back to a DNA duplex. sgRNA, dark blue; target...DNA strand, and Cas9 bends the DNA helix by transitioning between the open and closed conformations. This... spots—to these questions. Each new structural insight also spurs the next generation of engineered CRISPR...

...Addgene blog Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...experiment, read our blog post on important considerations when using AAVs. Experimental results: These...Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting using AAV on...

... genetic condition leaves patients incredibly sensitive to sunlight and prone to developing metastatic... into XP genes and other genes involved in photosensitivity in melanocytes with the goal of understanding...explore the functional differences among mutations responsible for the disease known as Alternating Hemiplegia...disorder.” In 2012, researchers identified the gene responsible for the disease as ATP1A3. The gene encodes a...

...next and what options lie ahead of me? Like a considerable number of researchers, I had high hopes of securing...thought they would focus on “Has this person been consistent throughout their career?”, "Have they sought ...interviewed for 1 hour so having a broad enough insight was going to help me push both my scientific and...promotions or further education may allow you to transition into various realms of business.  Thank you ...

...do you make the most of this new relationship? Consider adding formality and active goal setting to your...major advantage of working with a mentor.The responsibility of arranging meetings and setting the direction...meetings and discuss these in person. See the extensive resource list available from this post. Use an...items and make progress. To illustrate this point, consider that I try to exercise every morning, but the ...

...lab knows how expensive tiny amounts of reagents are, how complicated (and again, expensive) the different... college level, perhaps consider using some elements of BioBits to transition your undergraduate students...

...of the University of British Columbia. When transitioning from an academic science career path to a non-academic...applying for the position?   Here are a few things to consider when you're preparing your resume for a non-academic...postings and look at the jobs you are interested in. Consider listing the skills you already have, and the skills...Originally on track for a research career, she transitioned into a communications role instead after she...

...the plasmids we distribute. This method is time-intensive, so we recommend a variety of ways to screen and...10kb, with the 3.0kb fragment having increased intensity to serve as a reference band. The uncut DNA (lane...accurately visualize the full size of your construct. Consider buffer and temperature compatibility when digesting...methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity may be blocked...