We narrowed to 877 results for: TOR

... Set up a table of contents in the top directory: A directory is a file system in the computer and is...experiment its own directory: In addition to output files, this is also where you can store any power point...in C++ and Java Command line history: Tracking command line history by recording all commands entered...analysis like a wet lab experiment I use the text editor TextWrangler to record the following, which are...name for a folder. For instance, your Desktop directory contains the folders and files that you see on...on your desktop. The location of your master directory or folder for all of your analyses is up to you...README.txt document is invaluable. In the top directory of every experiment, immediately write a text ...

...employing optogenetic regulators to initiate the event being monitored. Optogenetic regulators respond to specific...fluorescent protein (acceptor) in the pair. The higher the emission from the acceptor, the more powerful...and NanoLuc® Luciferase are available from the repository. We encourage people to go to Addgene to get ...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse...applications of the Enhanced Nano-Lanterns including monitoring multiple cellular events such as the dynamics...initiation wavelengths, excitation of the optogenetic regulator leads to spurious activation of the FRET sensor...still transfer energy to the fluorescent protein acceptor. NLuc is an excellent BRET donor due to its bright...

...restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise the...use of both when making plasmids. Use repositories Repositories such as Addgene help share materials quickly...After the great Plasmid Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid...for the simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase.... This cost varies hugely based on a number of factors, but we can assume it’s at least equal to the $155...in a 96-well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any...any of the numbers in this post is a mathematical story well beyond my ability to tell. But even from these...

...knock out plasmids? Check out our Cre vector and Murine LoxP vector.   Turn it off for a little while Transient...? Check out our Retro expression vector and Lenti expression vector. Turn it on for just a little while...CRISPR for that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The...out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the time If ...Addgene's Lentiviral dTAG cloning vector and SMASh degron tagging vector! Destabilizing domains Destabilizing...increase expression. There are several flavors of activators which can be used with this approach - read more...them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently...

...begun to use more precise genetic tools: activators, inhibitors, sensors, recombinases, and more. With ...by using cis regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational...Addgene blog Download the viral vectors 101 eBook Read our viral vector related blog posts Learn more about...With the help of adeno-associated viral vectors (AAV), neuroscientists can combine classical genetic tools... how these changes affect the phenotype. Viral vectors and genetic approaches are continuously refined...Education and Development workshop aimed at brainstorming strategies for using combinations of AAV-based...designing experiments with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route...

... they monitor. Gene traps Gene traps are promotor-less constructs that contain splice acceptors. They...below), but fundamentally the reporter construct monitors the expression of a nearby gene. The reporter ...high/low) and patterns (tissue/timing) can be monitored under various conditions without necessarily modifying...stage you could infer that the gene which GFP was monitoring was being expressed in the same pattern. These...throughout an organism’s genome. These reporters have historically been used extensively in fly, mouse, and yeast...result but will still allow for expression pattern monitoring. Enhancer traps Like promoter traps, enhancer...traps, it will still likely disrupt the gene it monitors due to transcriptional fusion.   Examples...

... of stimulation to receptors. Optogenetics offers better control over these factors as the light source...more about optogenetics New to viral vectors? Browse our Viral Vectors 101 blog posts Learn more about precise...Addgene.org Browse all viral vectors available at Addgene Visit our viral vectors guide Find our chemogenetics...different advantages and limitations.  The four main factors in which chemogenetics and optogenetics differ ...offers a high degree of temporal resolution as receptors can be turned on and off simply with the presence...diffusion of drugs throughout the body, the onset of receptor activation takes minutes. As a result, it is not...response or track the exact time of activation. Receptor activity is also drawn out as drugs can remain...

...inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated...promoters. Other researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate enhancer...methylation and activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD, is available...and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal. Recently, Albert...alone, or coupled with DNA oxidation and repair factors that allow for increased gene activation at the... targeted enhancers. Although the lab used this vector in chick embryos, it also functions in mammalian...and this fusion allows for packaging into viral vectors and targeted delivery into hard-to-manipulate cell...

...bacterial prey. Currently, receptor binding proteins (RBPs, the host recognition factors of phages) constitute...Therefore, in addition to their utility in the health sector, phages represent a reservoir of carbohydrate binding...function of digestion time. For phage DNA that is refractory to enzymatic digestion, however, a physical fragmentation...a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing exist as ... blunt ends) and ligate into a bluntly digested vector designed for protein overexpression in E. coli,..., such as pET30a. If working with DNA that is refractory to enzymatic manipulation, amplifying phage DNA...primers bordering the multiple cloning site of the vector. In order to reduce the chances of some clones ...

...Existing methods for AAV quality control Historically, AAV vector QC has been sparse including only titration... procedures and Python scripts with laboratories and viral vector cores routinely producing AAV. We believe... our viral vectors related blog posts Read our blog post on AAV titers Browse viral vectors protocols ... platform to accommodate adeno-associated viral vector (AAV) samples and created a simplified process,... of quality control is sufficient but for viral vector core facilities producing a large number of viral...demonstrated that NGS is an effective tool to confirm AAV vector genome identity. These studies used SMRT sequencing... simplified approach, we sequenced over 100 AAV vector preparations and showed that VGS can quickly and...

...Louis provides a combinatorial indexing strategy that simultaneously tracks clonal history and cell identity...embryonic fibroblasts (MEFs) into induced endoderm progenitors (iEPs), a self-renewing cell type that can differentiate...iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like other reprogramming...CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), and 13 days... Morris Lab found that cells took one of two trajectories: a route towards successful iEP conversion or... The CellTags revealed that cells with common ancestors tended to have similar reprogramming outcomes....2019). Key takeaways of CellTag CellTag is a combinatory cell-indexing tool that combines scRNA-seq and...

...and money producing virus from select vectors in the repository yourself, you can now order ready-to-use...As some of the most popular lentiviral vectors in the repository are used to produce CRISPR tools, we’re...Expanding the viral vector knowledge base: You can help! We’ve been expanding our viral vector educational resources... production process while still ordering viral vectors. Virus production can be a frustrating process ...delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select gRNAs, and CRISPR pooled...expression of a gene under the control of the tet operator. AAV AAVs are the workhorses of both chemo- and... work with virus. You can find all of our viral vector blog posts here. If you have virus expertise you...

...posts related to viral vector protocols and tips Read all of our viral vectors blog posts Resources on...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ...titrating AAV by qPCR, viral DNA is amplified and monitored in real time. When the PCR is complete, the results...issue with qPCR is that the results can vary by a factor of 2. This means that if you set up two identical.... This partitioning reduces the number of PCR inhibitors per reaction and allows for enhanced detection...up with poor droplet quality. Using a droplet generator, the sample and the droplet generation oil are... = Volume of virus in reaction mix D = Dilution factor of virus Most droplet readers have a few channels...

... AAV vectors expressing RGB proteins under the control of an inducer, i.e. tet-off transactivator (tTA...Subscribe to Viral Vector Blog Posts Additional Resources on Addgene.org Search for AAV Vectors to use with...   Adeno-associated viral (AAV) vectors are the most frequently used gene-transfer tools in the study...infect the PNS or the CNS much better than the AAV vectors they were derived from. Additionally, AAV.PHP.eB... of AAV.PHP.eB and AAV.PHP.S to their parental vectors. To quantify this comparison, capsids were packed...AAV-PHP.eB and AAV-PHP.S versus their parental AAV vectors. What can these AAV’s be used for?  1. Multi-color...multi-color labeling is to inject a mix of AAV vectors that constitutively express a red, green, or blue...

... plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles. Today, it'... in this post:  New compact, efficient prime editors A versatile and precise system to improve on-target...proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old antibody...antibody new tricks   New compact, efficient prime editors By Susanna Stroik The Liu lab recently used phage-assisted...protein engineering to generate seven new prime editors, PE6a–g, that overcome some current prime limitations... and PE6b are 500–800 bp smaller than previous editors, but just as effective and with no additional off-target...protein engineering yield compact, efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell...

...the activator protein cannot bind. After an inducer binds to the activator protein, the activator protein...In this system, the activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive...the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription...agents, temperature, and light are all examples of factors that can lead to the induction of a promoter. Below...inducible system is found in many bacterial expression vectors. Negative inducible promoter pBad is another popular...protein purification. When arabinose is absent, regulatory protein AraC binds O and I1 sites upstream of...addition to arabinose, cAMP complexed with cAMP activator protein (CAP) can also stimulate AraC binding ...

...zinc finger proteins, and transcription activator-like effectors (TALEs), with fifteen total variants deposited... plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles. Here's what...systems for specific applications! Delivering prime editors (PEs) via engineered virus-like particles (eVLPs... contain no genetic material, which ensures the editor will only be transiently present in transduced ...site-specific serine recombinase. First, the prime editor installs the recombinase “landing sites,” then ...landing sites on opposite DNA strands. When the prime editor releases the DNA, the two 3’ flaps containing the...-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nature Biotechnology...

...G-protein-coated RABV infects cells via the G receptor. In neurons, the G receptor is restricted to presynaptic nerve...Control Gene Expression with FLEx Vectors Resources on Addgene.org AAV Vector for TVA Expression AAV for Rabies...housed in specific locations and give rise to progenitors during growth and development or that blood flows...to visualize how neurons are connected. Viral Vectors as Neuronal Tracers Due to their ability to efficiently...through their interactions with target cell surface receptors (Callaway 2008). Based on this, cell type specificity...EnvA-pseudotyped RABV, and only expressing the EnvA receptor (TVA) in user-defined cells. For example, a researcher... via entry mediated by the G protein/G-protein receptor. Once in first order neurons, the G-deleted RABV...

...acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens has written ...were many changes to the  Board of Directors and the Executive Director changed twice. I had to learn how... to report to the Board of Directors and liaise with the Executive Director. As members changed, I had...management-for-scientists-managing-vs-leading. The National Postdoctoral Association (NPA) outlines 6 Core Competencies... and Management skills”. The NPA states that Postdoctoral scholars should have the skills and techniques... levels. These skills will also help the person mentor others more successfully. But how does one gain... leadership experience). I was involved with postdoctoral issues first at Brigham and Women’s Hospital...

...the vector backbone you cloned your insert into and fill out the appropriate boxes with the vector backbone...backbone manufacturer, vector backbone size, total size of your vector + insert, any modifications you ...standard conditions (37 °C, LB, antibiotics, etc.) and stored as glycerol stocks We will need information on...provide any full or partial plasmid sequences and any vector maps. The more sequence data available, the better... was a part of the insert (when cloned into the vector backbone). If your plasmid has more than one insert...you made to the backbone, and the vector type. Pro Tip! Reference the Addgene ID in the "Backbone Manufacturer...enter "Feng Zhang (Addgene plasmid #52961)".   The vector type should describe how your plasmid is intended...