... cells are sensitive, it might be more difficult to get them to grow out. The only downside is that it.... In theory you could use densitometric analysis to quantitate the intensity of the bands on your gel ...reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective...In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively easy to transduce...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...followed. If you have never used this assay before, consider following their tips, including: Before digesting...

...or qPCR-based methods to greatly increase the sensitivity of detection (14-20). LDR has also been extended... do not read specific base sequences, but are sensitive to distortions in helix shape (22). Large purine... can be achieved through a combination of the intrinsic selectivity of the ligase active site and careful... that extends earlier studies through a high-sensitivity multiplexed format (21, 27). This methodology...In this method, substrate pools were prepared consisting of one target (template) strand and four upstream...Eukaryotic DNA ligases: structural and functional insights." Annu. Rev. Biochem. 77 (2008): 313-338. PubMed...JS, et al. "A high-throughput assay for the comprehensive profiling of DNA ligase fidelity." Nucleic acids...

...GCE4All: Making Genetic Code Expansion Accessible Addgene's Genetic Code Expansion Collection...protein tags. The basics These tags most often consist of an enzyme that has been mutated to block its...CP appearance at the filament tip from (a). c) Intensity traces at the tip of the filament from (a-b), ... effects on actin filament length (lower). d) Intensity and filament length traces for a different event...of organic dyes or specialized ligands is more expensive than letting the cells make a fluorescent protein...Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science, 281(5374), 269–272. https:... W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research, 44...

...turned to optogenetics. Through the use of light-sensitive protein domains, they have developed a system .... They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into a loop region ...brain. But this process is time consuming and expensive. Thus scientists have turned to genetically encoded...electrical activity via imaging. They took the light-sensitive protein QuasAr2 and systematically mutated it....localization, has high signal:noise ratio, high sensitivity, responds quickly, is photostable, and is compatible...interaction (PPI) studies in plants that rely on transient transfection systems using Agrobacterium (such...Grefen and Blatt, 2012). The 2in1 Plasmid Toolkit consists of 4 plasmid toolkits generated with 4 different...

...recently developed a third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3) to...and their research.   Abhi Aggarwal: Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate ...new version of the biosensor is faster and more sensitive than its predecessors. Currently, Aggarwal, and...Research Center (DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based biosensors for live cells.... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...

...within 10 nanometers of each other, or measure the tension in the helical structure of spider silk, or the...As the choice of fluorophore pairs is a key consideration for a FRET experiment, the possibilities can...Förster resonance energy transfer by monitoring sensitized emission in living plant cells." (2013). PubMed...linker sequence between the donor and acceptor is sensitive to a change in the environment which alters the...processes and signaling cascades, quantify mechanical tension (a molecular “spring”) (Meng et al., 2008), and...animals (Hirata and Kiyokawa 2016), and molecular tension microscopy (MTM), which could monitor physical ...Charlène, and Borghi, Nicolas. "FRET-based molecular tension microscopy." Methods 94 (2016): 33-42. PubMed PMID...

...for optogenetics. There are several important considerations with fabrication of the fiber optics needed...stimulation. The scatter effect is also an important consideration when preparing the implant itself, which brings...will be erratic, opsin activation/inactivation inconsistent, and behavioral results impossible to interpret...sequentially increasing fineness helps to maintain a consistent stream of light. Test your implant by connecting...least 85% of the initial laser output. The same considerations apply to the polishing and preparation of the...: closed/light receptive, open, closed/light insensitive. If you deliver a continuous stream of photons...optogenetics we’ll delve into some of the important considerations when performing optogenetics behavioral experiments...

...methodological and experimental limitations and considerations? I recently spent two months in the lab of ... at NIDA and some of the practicalities and considerations you may not find in the materials and methods...use optogenetics The most important question to consider is what exactly do you want to use optogenetics...are also available. Picking your opsin  You must consider whether you wish to stimulate or inhibit neuronal...include Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation of which...potential. Halorhodopsins (HR) are green/yellow-light sensitive chloride ion pumps, activation of which results...breeding, and maintenance of a mouse line is also an expensive and time-consuming endeavor. Finally, expression...

...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...complexes with. These “colocalization” studies give insights about the proximity of two proteins within the...cameras and image analysis software are highly sensitive to detecting light signals not visible to the ...human eye and are indifferent to subjective color intensity perception. Open source image analysis software... and well-executed experiments require some consideration of FP properties. It is important to look out...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...

...such as mice, which are less prolific and more expensive per individual to maintain. Also in contrast to...heart defects and cancer. These mutants are used extensively to study development and disease. Additional ...implemented in zebrafish. Developmental biology insights using lineage tracing in zebrafish To highlight...lineage tracing approaches that led to important insights in developmental biology. Microscopy-based lineage...embryo, where similar processes occur over days inside the mother, the zebrafish is an ideal system to...their long three-month generation time and labor-intensive maintenance compared to invertebrate model systems...highlight how these advantages can translate into insights in developmental biology, and advances in this...

... as one of the the skills favored by research-intensive careers. External collaborations are becoming ...important in a non-research context, especially when considering small to mid-sized companies and organizations... Addgene as a nonprofit organization is very considerate and thoughtful about its limited resources. Rather...find this to be the most challenging and time-intensive part of relationship building. However, it is ...without directly contacting them. One great way to transition from an internal to an external connection is...organization is important for success in research-intensive careers. In addition, this skill is a hallmark...Collaborate: An Imperative for Graduate Students" from Inside Higher Ed Additional Resources on the Addgene Blog...

...library/produced your lentivirus. Since viruses are sensitive to freeze/thaw cycles, we include a 1.3 mL aliquot... number of cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing...transducing cells at a concentrated seeding density while centrifuging. Spinfection transduction efficiency...optimization of many variables, such as seeding density and reagent concentration.  Screening time Phew...place to ask experimental questions.  Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...Libraries- A nice overview of what to consider when using pooled lentiviral CRISPR libraries. Protocols from... Waxmonsky NC, Swanson LE, Fan M. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

... the end of this post! Figure 1: Base transitions are edits between the purine bases (adenine, ...newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar...transversion editor AYBEv3. This tool still retained considerable A → G editing activity at most sites, in addition... editor (gGBE) (Tong, Liu, et al., 2023). gGBE consists only of engineered MPG fused to nCas9. First, ...efficiency, purity, and indel rate of each tool vary considerably. And there’s so much more exciting protein engineering...is which. Acronym Full name Notes   Base transition Base change between purines (A ↔ G) or between... glycosylase to a CBE; the first type of base transition editor invented.   Abasic site A DNA site with...

...can be a time-intensive endeavor. That is why we also included an analogous set of transient constitutively-expressed...on the other hand, is a widely available and inexpensive imaging modality with good tissue penetration... be easily expressed heterologously by bacteria inside the body. So we screened gas vesicles gene clusters...bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the tumors was overlaid with the anatomical B-mode...Emerald GFP. These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293... using the most reliable method, which is the transient cotransfection of the mARG plamsids into HEK293T...

...” in quotations here because I would not have considered myself neutral in any of their disagreements....Skills series, this post is not meant to be a comprehensive guide to Conflict Resolution. There are countless..... Ownership: Misunderstandings about who is responsible for what tasks or projects is a common source...customer support element amongst their other responsibilities. Knowing which aspects of customer support... require a manager to explicitly assign the responsibility in question to a specific person or team. ... more of a winner and loser–one party will be considered in the right, and one in the wrong. If you arbitrate...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...

...primary antibodies is a time consuming and expensive process. Consider the vast number of possible primary antibodies...we’ll go through some of the most important considerations to make when planning and performing an IF ...indirect immunofluorescence procedure with key considerations about each step. Image created with BioRender.com...’ll start there. Some of the most important considerations are to make sure your secondary 1) will recognize...remember that antibodies recognize very specific 3-dimensional epitopes. Different sample processing steps can...protocols available for each antibody that you consider in order to determine how reliable the antibody...

... imaging NIR FPs, consider using light sources that produce enough light intensities in NIR. Usually, ...supplied (Baloban et al., 2017).  It is important to consider effective brightness of NIR FPs in cells, in addition...Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs were developed...dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with no supply of...Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. Chem ... (2015). Piatkevich, K.D. et al. A robotic multidimensional directed evolution approach applied to fluorescent...

...results of cloning or knockout experiments to forensics and clinical diagnostics. One example of southern... a sample. Whereas Southern blotting is labor intensive and requires a large amount of high-quality DNA...lacking the 3’ hydroxyl group required for the extension of a DNA strand (8). Adding a small amount of ...of DNA strands of all possible lengths because extension is terminated upon the incorporation of a ddNTP...speed ultimately leading to what are generally considered first generation DNA sequencing machines (9).... drive for faster, higher throughput and less expensive sequencing fuels a sequencing revolution that ...1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new prenatal test for sickle-cell anemia." New...

..., for some applications, integration might be considered a double-edged sword with unintended consequences...AKA transduction in the viral vector world). Once inside the host cell, the viral payload is integrated ...lentiviruses lack a functioning integrase, the enzyme responsible for inserting viral DNA into the host genome....genomes are transcriptionally active, leading to transient expression of gene products (Wu et al., 2004)....insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your...expressed (Fu et al., 2013). Using IDLVs ensures transient expression without the risks associated with prolonged...of clinical or basic research applications. Considerations for IDLV use If you’re reading this and thinking...

...include a Packaged on Request option. Through this expansion, scientists will be able to request an AAV composed... vector prep.    Excited? So are we! This expansion will give researchers increased flexibility when...overview in this blog post.     When will the expansion happen?  You will be able to request Packaged ... Plasmids available for Packaged on Request consideration will have a banner on their material page. Interested... in-stock preps. If a request has technical considerations that may affect expected titers, a customer...resources.  We hope you’re as excited about this expansion as we are! Happy packaging! ...