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... embryonic stem cell stage using one of several techniques. The longest standing technique is the injection...Mice are a common model organism used to understand mammalian traits and genetically engineered mouse... to create engineered mouse models at embryonic stages, and the pros and cons of different genome editing...experiments. Inbred mice are particularly useful for understanding a specific effect related to an experimental...positive selection marker such as an antibiotic resistance gene is also included to select for successfully...which is then implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks...which is then implanted into a female mouse for gestation. All three can introduce multiple base pair deletions...

...The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected...to move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure...transductions should not be rushed into. Before you start any transduction, you should always titer your virus...components such as LTRs, gag, WPRE, antibiotic resistance-genes, or the transgene itself. Many researchers...are used as it eliminates the need for antibody staining. Check Out Our In-Depth Fluoresence Titering Protocol...Another popular approach utilizes the antibiotic resistance genes carried on transfer plasmids. Target cells... one copy integrates, selecting by antibiotic-resistance or single cell sorting based on expression of...

...significantly higher than standard “wired” optogenetics but may become the standard in the future. Now that...by guest blogger Derek Simon. The surgeries and standard molecular neuroscience validation experiments ...Light is able to penetrate brain tissue a short distance so ideal implant placement should be about 0.4...significantly increases. Before you get your experiment started, you will need a laser of the appropriate wavelength...channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive, open, closed/light insensitive...light must be delivered in pulses. An example of a standard stimulation parameter is 20Hz (laser output), ...source itself. Pulse generation (TTL pulse) is a standard electronics protocol not specific to lasers or...

... to all our #DeckTheLab contestants!           View this post on Instagram                     A...December 12, 2022          View this post on Instagram                     A post shared by 草方 (@gracie_fang...take photos, and then share them on Twitter or Instagram with #DeckTheLab and tagging us, @addgene to be...get you out of.           View this post on Instagram                     A post shared by Xi Chen...picture or video for either contest on Twitter or Instagram by December 31, 2022.  Follow @Addgene, use the...and reveal the winning entries on Twitter and Instagram! Happy decorating, all, and to all a good light...

...ready to store or use. They are quite stable and will withstand freeze/thaw cycles and dehydration (Howard...you can also generate rAAVs in your own lab with standard molecular biology tools and tissue culture experience...choose the optimal titering assay, you’ll need to understand how different titers are measured. A TCID50 measurement...method due to the accessibility of instruments in standard labs. When determining titers by qPCR, we recommend... You should always have a high-quality plasmid standard and rAAV reference material of a known titer on..., D. B., & Harvey, B. K. (2017). Assaying the Stability and Inactivation of AAV Serotype 1 Vectors. Human...recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21(10), Article ...

...single stained reference controls to distinguish one fluorophore from another within a fully stained sample...called autofluorescence (you can see this in your unstained flow controls). Spectral flow cytometers include...tissue types varies, it is critical to include an unstained sample for each tissue type you are running in...Compensation or “reference” controls are samples stained with a single fluorophore. They’re required for...look for that exact signature within your fully stained samples. If you are using conjugated dyes, it is...particular difficulty in unmixing if your sample is stained with a PE/Cy7 antibody from a batch that is not...unmixing. Additionally, it is critical to include unstained beads or cells within your reference group to ...

...because it has the advantage of starting out with a larger initial starting library size (up to 1012 members...challenging when trying to develop antibodies against unstable targets like membrane proteins that may unfold...This selection platform was designed so that any standard lab can quickly select for sybodies targeting ...To find an optimally binding sybody, you want to start out with a large and diverse pool of sybodies that...al., 2018). Find the Sybody Generation Toolbox! Starting with libraries and a biotinylated (for purification...et al., 2018. Ribosome display The Seeger lab starts with a “pre-enrichment” selection step using ribosome...Zimmermann et al., 2020). This step of the protocol starts out with anti-Myc antibodies bound to an ELISA ...

...customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9 proteins....range of bacteria. Once transferred, Mobile-CRISPRi stably integrates into the genome and is propagated during...Mobile-CRISPRi does not disrupt gene function and is stable and active for greater than 50 generations. ...40-fold across all species measured. Beyond understanding the basic biology of bacteria, Mobile-CRISPRi...Mobile-CRISPRi can be used to identify antibiotic resistance genes. Genes that are targets of antibiotics are often... guides targeting non-essential genes remained constant, with fitness effects more pronounced at 12 doublings...dCas9. Following integration, Mobile-CRISPRi is stably expressed with no selection required and titratable...

...functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease...NgAgo is faring with users in a minute, but, to start, let's review why NgAgo is in the spotlight and ...difficult” GC-rich regions of the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and...sequence might not always be available at an optimal distance from the target site. For example, NGG may not...genome editing applications. So, how does NgAgo stack up thus far? A few weeks ago, I was going through...and fussy to optimize. One reason could be the instability of the supposedly alien 5’-phosphorylated ssDNA...optimizing genome-editing methods in brain cells and understanding brain tumor biology. When not snipping DNA, ...

...heavy chain, light chain, constant region, variable region   The constant region of the heavy chain..., or antigen-binding region, and the Fc, or crystallizable region. Two immunoglobulin (Ig) heavy chains...by changing certain amino acids to improve its stability. Additionally, the choice of vector can impact... your sample, you’ll compare your samples to a standard curve. This curve is generated using a serial ...protein that is also detected in the same way. The standard curve that is created from the OD and concentration...antibodies to detect proteins is referred to as staining. This panel of antibodies is used to both identify...Similar to flow cytometry, you’ll use antibodies to stain for markers of one or more specific cell types, ...

... So what’s a researcher to do if they want to understand the ultrastructural relationships that influence...expansion.  In some cases, labels are added after this stage.  Step 4: Expand! (Typically by just adding water...with biorender.com Steps 2-4 are not found in standard IHC/ICC protocols and you might expect them to...common reagents, straightforward protocols, and standard microscopes sounds great, right? But there is ...post-expansion, but that doesn’t mean we should start re-conceptualizing actin filaments or microtubules...scene, ExM has helped researchers gain a deeper understanding of neuronal synapses, centrosomes, parasites... Royal Soc 380:20210110. https://doi.org/10.1098/rsta.2021.0110  Sherry DM, Stiles MA (2022) Improved ...

...linearized plasmid standard. However, even when the standard is made properly, it is unstable and may only ...the viral DNA to the threshold fluorescence of a standard - a set of reactions with known quantities of ...a known titer, can be used to confirm that the standard curve is giving an accurate readout of your samples... range. We have also found that generating the standard curve for qPCR can be challenging to get right...Droplet digital PCR (ddPCR) does not require a standard or a reference, meaning saving on reagents (and...to contamination. Optimize your PCR For a good starting place on what PCR parameters to use, see Lock’...

...-person events and venues will have a solid understanding of their audience demographics. Do your research...can vary depending on factors like socioeconomic status and geographic location. For example, not all social...Before you embark on a project, do your best to understand what kind of resources - including your time ...specialized equipment and/or expertise from the start, or if you can begin simply and build up to more...potential of your preferred platform before you start can save a lot of trouble in the long run.  Selecting... your audience. Digital or print distribution.  Static. Can be difficult to convey an entire story or ...omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific training...

...Chemical fluorophores can be substantially brighter and more photostable than fluorescent proteins, providing... azetidine rings could substantially enhance the brightness and photostability of the classic rhodamines...enhance small molecule fluorogens (Figure 1e). Stains – These labels consist of a fluorophores conjugated...classic, net-neutral dye scaffold due to its established chemistry, small size, brightness, and cell permeability...In past decades, further refinements to this established dye structure have yielded commercial panels ...Fluor and ATTO dyes, with improved brightness, photostability, and spectral range. Nevertheless, these fluorophores...

...plate. You can use this plate to start liquid cultures. Lastly, you can start small overnight liquid cultures...detect the presence of your insert; 2) set up a standard PCR reaction (primers, dNTPs, polymerase) using...PCR reactions is nearly identical to preparing a standard PCR reaction: combine template, primers, polymerase...polymerase, and dNTPs and then incubate with a standard PCR thermocycling program. One key difference is ...leftover bacteria-water suspension and use them to start cultures of your positive clones. If you want to...the initial heating step of the PCR reaction. A standard Taq polymerase is sufficient. Controls: Controls...Make sure to use an appropriate molecular weight standard for reference and to add a loading dye with glycerol...

...Background on neuronal tracing A key aspect to understanding the brain’s function is knowing its architecture...importantly, any connections the neurons make to other distant brain areas. It is desirable for tracing compounds...the direction of information flow in order to understand which brain regions communicate with each other...decades was essential to build up our current understanding of neuroanatomy and neural connectivity, providing...advantage of AAV vectors is that there are well-established protocols for their production and purification...expressed a fluorescent marker in areas close or distal to the injection site across the entire brain, ...connectome”, to be visualized in a uniform and standardized way (Oh et al., 2014). Images yielded from this...

...to maintaining high QC standards for all deposited materials. The QC team stays informed of emerging technologies... process and therefore put in a lot of effort to stay up-to-date with these improvements. In this post...technologies are used, and how Addgene keeps up with the constantly updating technologies. Sequencing technologies... Sanger sequencing was considered the gold standard until about 2008 when a new, higher-throughput...high-throughput compatibility. However, there are some instances where we use long-read sequencing or Sanger sequencing...the assembly. Keeping up the pace Our QC team constantly faces the challenge of keeping up with the large... of QC analysis required every week. They are constantly exploring ways to enhance their processes, quality...

...choices you make during the early stages of the process. Some questions to start with are: Is your sequence...final vector structure you want to achieve before starting your experiments. Don’t dive directly into the...asymmetric DNA sequences and cleave at a short distance from their recognition sequence. While developing...learning about MoClo in an in-depth and easy-to-understand guided manner. These videos can be especially...perform your MoClo assemblies in silico before you start doing your cloning. Most sequence analysis software... the line! Cash in on gene synthesis Before you start your cloning, it is advisable to remove unwanted...glycerol stocks. It is very important that these stay frozen at -80 °C, since multiple rounds of freezing...

...regulations, differing customs processes, and the distances the packages needed to travel meant the accessibility...using couriers that refill dry ice if the package stays in transit for a certain number of days and tracking...Addgene distributors Distributor Partnership Start Date Country Served Beijing Zhongyuan 2012...information about the materials in our repository. We started to increase accessibility to our repository by ...types, and more, all of which help a researcher understand if a material is right for their experiment.   ...introduced guides — practical primers to help users get started with tools like CRISPR, lentivirus, and optogenetics...darker blue indicates more views from that country. Stats and figures are helpful, but our favorite measure...

... for numerous non-academic careers. Still, many start their scientific careers hoping to follow the traditional...Possible roadblocks only become obvious at rather late stages (i.e. postdoc level) and can include the pressure...candidates are highly experienced but often have to start pursuing other options. While some non-academic ...existed, but did not consider them for myself. I started picturing myself in a technical support role only...possibly this same reason, it can be one of the more constant and safe corners in a company, less exposed to...where someone tries to “optimize” a particular standard lab protocol. For example, someone may attempt...then complain about high background. In many instances, variations of the product’s specific application...