We narrowed to 888 results for: URE

... the off-target activity. Figure 2: Specificity is the measure of off-target activity (left) while...sensitivity. Site accessibility Site accessibility is a measure of which genomic sequences can be targeted by the... sites that were previously unavailable. Figure 1: The PAM sequence that the Cas nuclease recognizes...closely related to specificity. Sensitivity is a measure of the on-target activity of the Cas9 nuclease....frequency at a target site. A specificity ratio can be measured by calculating the on-target activity divided ...while sensitivity is the measure of on-target activity (right). Cas9 nucleases that exhibit enhanced...NGAN, NGNG, NGAG, and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9...

... adjustments I wanted to make to ensure smooth filming in the future, but we were finally refining the...I could be happy with it. Cycle 5 - The Future! Figure 5: The mobile monitor and teleprompter being...trying all over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for...anticipated, we had to think of something else.   Figure 2: Ironing boards. Good as a WFH desk, and as a...important for me to let people record at their leisure. For virtual B-Roll (that’s the secondary footage...drawing board.  Cycle 3: A Return to HQ Figure 3: Even Blugene is practicing social distancing... left the room. Microphone: Lastly, I needed to figure out a microphone situation. Generally, I record...

...extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction (or...overlap ensures that the PCR generates a nicked circle rather than a linear product (see figure). Template... simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust the extension time to ...vivo to give the final circular plasmid product pictured here. B) Screening of recovered plasmids for the...final concentration). The DMSO reduces secondary structures of the DNA template, and may also decrease the...template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired mutations through...polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases...

...Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr...some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello G... mouse model, an important proof of concept for future preclinical and clinical work. Find Plasmids for... is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV ...homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping proceeds...would be applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular...low-frequency indels will not cause frameshifts. Figure adapted from Osterout et al. (2015b) under a CC-BY...

... like pipets, or mini-centrifuges, etc. Be sure to capture as many of these tools as possible, this even... we suggest estimating the time it will take to secure your first (R-level) research grant.  Set your ...down prices as you create your list. This helps to ensure that all of the resources are accounted for. Write...an idea of how much you can expect to use in the future. 3. Identify the skills needed to use these tools...the talent pool that you’ll need to tap for your future research team. For example, if you’re going to ...help you define salary costs. 4. Ascribe a cost structure associated to these tools, skills, and personnel...searching for a faculty position. This is important to ensure that you’re looking at institutes that have the...

...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can ... which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial...implantation of fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains...improved photocurrent properties: The sequence and structures that determine membrane expression and plasma... electrophysiology, a technique that’s used to measure electric current of ion channels in living cells...electrophysiology. All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity...were then characterized further by expression in cultured neurons and mouse brain slices collected following...

...to define what features would allow for the best performance in the most contexts (Figure 1). Together ...quickly losing activity. I set out to collect the features that would define a good tool for sticking together...Together the features with optimal biological performance are described as Click Biology (Howarth, 2025). ... Figure 1: Modular assembly tools, alongside criteria for Click Biology. Created with BioRender.com...bonds. Reactions also can happen at both body temperature and on ice, when we have cracked open the cells...genetically engineered components, like protein tags (Figure 2). Through the work of many groups, there are ...found in Addgene's repository (Table 1). Figure 2: Adding function to My Favorite Protein (MFP)...

... Design and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), ...Interactions between the target strand and the gRNA (see figure below) Because off-target sequences have less ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to ...improve the specificity of Cas9 derived from S. aureus. This means you can likely apply similar rational...of genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers..., Chen et al. 2017 used single molecule FRET measurements to determine which portions of Cas9 might be...HypaCas9 plasmids are available from Addgene here! Future possibilities for Cas proteins With their enhanced...

...p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in...plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters...also available as ready-to-use lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters...LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC...silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation persisted ...period, as this mark was faithfully propagated in culture and in vivo. In certain cases, epigenetic modifiers...acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517 . https://doi.org/10.1038...

...experiments require positive and negative controls to ensure proper interpretation of results. A positive control... to compare the experimental group.     Figure 1: A, Most biological experiments will benefit ...group and C, technical replicates or repeated measurements will increase the power of the results. In this...experiment. Technical replicates are repeated measurements of the same sample and are included to assess...long as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should...molecules through the interactions of other antibody structures, such as the Fc region. Fc-mediated binding interactions...as with an isotype-matched control.      Figure 3: A preimmune serum control addresses situations...

...bacterial culture into the late stationary phase. Growing the culture at a lower temperature such as 30...  Figure 3. Summary of plasmid multimerization troubleshooting tips. Portions of this figure were ... More research is ongoing to determine plasmid features that affect the frequency of multimerization. .... Theoretically, as long as all the plasmid features remain intact, plasmid multimers function just as...recA-independent mechanisms. Avoid overgrowing the bacterial culture Another way to prevent plasmid multimerization ...optimizing the incubation time can help maintain cultures in log phase growth. In a study by Williams et...plasmid can exist in a bacterial population as a mixture of monomers and various forms of multimers (Summers...

...polysaccharide structures, many of which are easily varied by the cell and whose structures are just beginning...around 8-12 h at room temperature as opposed to overnight at optimal growth temperature. If you’re lucky enough...traction lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell... for by manipulating the nebulization time and pressure. Nebulization units, which can be purchased cheaply...attached with a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to subject your membrane...

...everything was more structured and controlled. I worked from Standard Operating Procedures (SOP’s) and every...perfect duration for me because it was sufficient to figure out if my image of lab work lived up to reality..., but in research you also experience a lot of failure and problem solving. I believe that is something...they never really prepare you for the amount of failure and problem solving scientists experience during...in ways that people from another background or culture could understand. I did, however, find it very ...learn how to cope with a different climate and culture, and also needed to be resourceful and adapt. By...doing experiments  without the constant fear of failure because I knew the tests worked and would produce...

...My name is Lauren Flynn and I am currently wrapping up my co-op with Addgene as a DNA Sequencing Analyst... member of the Quality Control team, I help make sure the plasmids that go out our doors are correct. ...minor, it’s time for me to start considering my future path. Talking to Addgenies has helped me get a ...concepts that are heard often during undergrad. Lauren Flynn is the Addgenie behind our "Meet an Addgenie...years ago at a CRISPR meeting.Read his Instagram feature here. You also don’t have to go back to school...that if you don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize...with our resident dinosaur! Read her Instagram feature here. One of our QC Scientists, Shreya Vedantam...

...through the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed...difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a... to knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert...for targeting constructs. In the example shown in Figure 1, large sequences corresponding to exons 1 and...-eGFP-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed... and the targeted exon in the gene of interest (Figure 4). When recombination occurs, the gene still functions...cassette has been placed into an intron.    Figure 4: A targeting vector to create a floxed allele...

...long dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire...involved in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing... modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically...that facilitates the formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi...greatest advantage of RNAi is the straightforward nature of the technique—all that is required dsRNA complementary...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be ...

...fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 ...domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer a series of "split" cas9...for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs ...light-mediated subcellular localization in mammalian cell culture and reversible control of signalling pathways. ... off and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant....g. the ER) and a reporter protein of interest (Figure A). Franck Perez’s lab has engineered hook proteins...

...halt degradative processes while maintaining the structure of your samples, the relationships between cellular...that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...cross-linking, but it doesn’t preserve sample structure quite as well as a cross-linker. This is because...fixation with these chemicals, which impacts sample structure and could also cause your target to be washed ...Furthermore, these chemicals can also alter the structure of proteins, which could disrupt your target epitope...good thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...dehydrates the protein, which can disrupt protein structure and thus structural epitopes, but can reveal linear...

...vectors effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used ... you can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral particle, which...   Figure 3: The life cycle of a retrovirus. Image courtesy of Jeremy Luban, MD. Figure 3 depicts...vectors correctly. A solid understanding of the structure of a wild-type viral particle — and of the normal...depicts the viral life cycle. One should ponder this figure often, especially the three steps from the 12 o...

...bacterial recombination pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr ...hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or purification...be immediately adopted by any laboratory. All procedures are performed using the same three step protocol...cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid...design for each type of modification is shown in Figure 2, and is discussed in more detail below. The main... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...assembly. Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple...