...This method utilizes a packaging cell line that stably expresses Gag-Pol and/or Env, eliminating the need...packaging cell line developed by Garry Nolan at Stanford (Swift, 2001) contains Gag-Pol and either an ecotropic...differentiated (non-dividing) cells. Both lentiviruses and standard γ-retroviruses use the Gag, Pol, and Env genes...provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition of a poly A tract just...

...phenotypes than standard RNAi protocols without the need for mutant genetic backgrounds. The standard RNAi vector...biochemical and biophysical study involving protein stability or binding experiments. Potential experiments ...perform using the purified domains include protein stability, small molecule binding screens, peptide arrays...

...for colonies that contain your plasmid is a great start when cloning, but how about being able to choose..., and remain the lovely whitish-cream color of standard E. coli. On the other hand, intact β-galactosidase...colonies that not only have your plasmid (antibiotic resistance), but also confirm those plasmids have your insert...plasmid-containing cells without using antibiotic resistance. These rely on cell lines susceptible to or dependent...

...the problem and gather information to help you understand it. Step Two: Come up with and evaluate potential...You might do some background research to better understand it and come up with several hypotheses on the...problem solving process on your own, but you can start by paying attention to how your mentors reach solutions...to remember that you have these skills when you start applying and interviewing for new positions. Highlighting...

...will enable significant steps forward in our understanding of synaptic and circuit functions in health ...metabolites are vital for researchers to better understand the processes that happen inside the cell,” says...Together, this project will allow us to better understand how neuron activity changes following opioid ...improved voltage indicator coming from the screen to understand mouse cortical and hippocampal neural microcircuits...

...interfere with fused protein function Under some circumstances, the fused fluorescent proteins can be a roadblock...that some fluorescent proteins can be, in some circumstances, detrimental to the expression of the protein...be beneficial in some types of experiments (for instance, when the fluorescence expressed in the neuropil...techniques, her research aims to gain a deeper understanding of the physiology and pathology of the basal...

...weeks is the standard ballpark estimation. Once expressed, viral cargo is usually quite stable and in some...and promoter) for use in your experiment! As a starting point, we recommend checking the literature and...272. PubMed PMID: 12349826. Xiong, Wenjun, and Constance Cepko. "Distinct expression patterns of AAV8 vectors...

...protein degradation due to other pathways, it is standard practice to compare degradation rates between ...identify because their contents can be at various stages of degradation. Additionally, electron microscopy...that affect autophagy (ATG) in yeast. Atg8p (“p” stands for protein) associates with autophagosomal membranes...green fluorescence. After 4 hours of nitrogen starvation to induce autophagy, red but not green fluorescence...

...ratio, high sensitivity, responds quickly, is photostable, and is compatible with optogenetic control. ...proteins that bind to either the light- and dark- states of the photoswitchable cPYP, which changes in conformation... partners that bind to only the light- or dark- state of the photoswitchable proteins offers a powerful...binders (which bind to either the light- or dark- state of the photoswitchable proteins) fused to mVenus...

... of replication, protein tags, and antibiotic resistance markers (just to name a few). In this edition... I use Cre-lox? The Cre/lox system is a well-established research tool, especially in the field of mouse...targeting, targeted clones are selected for using a resistance marker; however, it is often desirable to remove...in certain cell or tissue types, during certain stages of development, or by making the Cre inducible ...

...chloramphenicol resistance pCRISPR: carries a spacer targeting the gene of interest and kanamycin resistance E. ...strand, among other parameters. .  Stanley Qi lab's CRISPRi plasmids Stanley Qi’s lab demonstrated that a minimal... specific polymorphisms, including antibiotic resistance genes. This approach successfully targeted specific...

...scientists have developed calcium sensors to better understand how this metal ion is trafficked and regulated...selecting a sensor to use for your studies, you can start by asking ‘What is the biological phenomena I want... sensor expression if you aren’t working with a stably integrated biosensor. Ratiometric measurements ...switches from green fluorescence to a red fluorescent state only by simultaneous (1) user-controlled illumination...

...fidelity. NEBridge GetSet Need a set of overhangs, stat? GetSet can do that for you, complete with predicted... °C cycling 4-base Bsal-HFv2® 37 °C Static 4-base Bsal-HFv2, 1x NEBridge Ligase MM ...They first compared 10-piece linear (the native state of the T7 genome) and circular assemblies. While... complex Golden Gate Assembly, a great place to start is the 24-piece lac operon assembly deposited by...

...widely used base editing systems, ABE7.10 (and the starting point for a new generation of ABEs), consists ... needed to produce 3 concurrent base edits to withstand antibiotic selection. As a result, 8 mutations... which resulted in variants named “ABE8.x”: “8” stands for the eighth round of ABE evolution and the “...frequency over ABE7.10. The ability to use non-standard Cas9 in the ABE8 complexes enables a broader targeting...

...AdV plasmid construct from step #1 and allowed to stay in culture for up to 20 additional days at which...is required if rAdV is to be used in vivo. The standard method for purification of rAdV uses cesium chloride...viral DNA packaged in virions is determined by a standard curve of known quantity and primers specific for...with serial dilutions of rAdV, fixed, and then stained 10 days later. Plaques are counted and titer is...

... locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters to knock... of exons. Disrupting promoter or transcription start site function would also help to reveal the impacts...polyadenylation signal downstream of the transcription start site will terminate transcription of most of the...based approaches to study ncRNAs. (A) Transcription start site (TSS) deletion. (B) Mutations of splice sites...

...review analyzing and explaining the antibody crisis states that up to 50% of all commercially available antibodies... to have an intuitive guide for checking their staining and neuroanatomy without the fear of referencing...please contact us here! Ava Shipman is a research assistant in lab of Zhuhao Wu at the Icahn School of Medicine...Bradbury, A., Plückthun, A. Reproducibility: Standardize antibodies used in research. Nature 518, 27–29...

...in helping us understand the nature of your concern. Please be courteous. We understand how exasperating...ready. I've attempted to answer several emails that start: “Hi! We ordered something from you and it’s wrong...last digit: An answer should come soon, but not instantly. Addgene tries to get back to customers within...

...in vivo studies, our scientists perform all the standard quality control assays for AAVs. These steps include... Unfortunately, humans have been known to make mistakes. What if a tube is mislabeled or someone grabs...vectors? Viral genome sequencing Thanks to the establishment of in-house sequencing capabilities and Seqwell...sample at 5%. Detecting 5% contamination is a good start, but to determine the sensitivity of our method ...

...cell, organelle, and protein localization. For instance, you might dye your cells and look at them under...researchers can look at a single image of a cell and understand how protein localization changes over time.Hallmarks...can be used in any situation where one wants to understand the relationship between the age of a cell, protein...biological event (trafficking to a subcellular location, start of gene expression, development of a cell structure...