...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...

...might not be a big factor for you. Know who's boss You sign on to a lab for the mentoring only to find out...choose a mentor that sees you only as a paper making machine. Choose one that is a true mentor, supporting...rotation was with a mid-sized lab with an experienced mentor. The lab was very, very quiet. Every time I asked...some of these questions in the sections below. Mentoring relationships can help identify your priorities...not worked for you in the past. For all of the factors I touch on below, the first question to ask yourself...“which ones matter to me?”  and hone in on the factors that will make the most difference for your success...? Will a postdoc or lab manager be your actual mentor? Is there a chance the supervisor will be moving...

...him a more effective motivator (“be like Tony Robbins!” he encourages faculty mentors.)  Collaboration Maximizes...technical and pedagogical resources, offers mentoring for instructors, and provides opportunities for students...in a year-long biochemistry laboratory : Part II – research-based laboratory – a semester-long research...becoming just the fifth repeat Nobel Prize winner in history. Interestingly, he also adds to the disproportionately...this trend as evidence that the best teachers and mentors pass on more than just technical knowledge, shaping...responsibilities left him with limited time for one-on-one mentoring. His solution? Bringing his research into the ...work identifying new genes required for stem and progenitor cell maintenance in planarians in Developmental...

...has a great team of moderators who facilitated scheduling, filtered out inflammatory questions, and even...and the moderators were on top of any comments that were at all trollish. Kudos to the moderators for keeping...trigger phrase these days). Redditors ask serious questions What the moderators did not delete or suppress...incredibly important to find good mentors and incentivize good mentorship: AMA mechanics: Be prepared! The...Addgene Executive director, Joanne Kamens, recently participated in a Reddit AMA (short for “Ask Me Anything...lists of forums on particular topics. Volunteer moderators set the rules for these forums and try to keep...careers, women in STEM, work-life negotiation, and mentorship, we decided to have her participate in an AMA...

...empty control vector would be Plasmid A sans shRNA, or backbone Y alone. The empty vector control allows... In this context, the empty vector control shows any effect of the vector/backbone itself on gene expression...hypothesis: proper controls! For every experiment, an investigator needs a standard against which the results can...variable you are testing and not some other unintended factor. Control plasmids are used to minimize any effects...planning your experiment includes determining what factors need to be controlled for in order to eliminate...replicate controls.  Transfection controls: empty vector and internal control A transfection control measures...any effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or ...

...few inventory items offered domestically, but by the end of 2016, we expanded our viral inventory to 25... is a dual-vector system, meaning it is typically used in conjunction with a second vector that delivers...constructs (i.e., using a dual-vector system as opposed to a single-vector system) has several benefits.... lentiCas9-Blast Most of Addgene’s lentiviral inventory is based on CRISPR tools, though we do provide...LentiCas9-blast LentiCas9-blast is a Cas9 expression vector developed by Feng Zhang’s lab at the Broad Institute...such as lentiGuide-Puro. The alternative single-vector systems typically express Cas9 and a gRNA from ...compared to separate transductions with a single-vector system). Tips for using this plasmid Cas9 expression...

... 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 ...

...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...

... 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...

... 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...

...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...

... you want the works: organizing reagent storage and inventory, managing orders, tracking students and ....io beta platform for sharing, annotating, and storing life science protocols. Since we began sharing ...website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the Broad Institute...Institute) has consistently been one of the repository’s most frequently requested plasmids. I wondered how many... and you decide to share them using Addgene’s repository, you can also share your associated protocols...listed under “Supplemental Document”: gRNA_Cloning Vector (deposited by George Church’s lab). Addgene has...the software aims to be a life science protocol repository. The software can be used with the ZappyLabs ...