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...first question is: to screen or to select? Which method will work best for your scientific question? If ...an assay to look at an outcome. Your scientific question can help determine which is right for you. Screen...interest is highly specific to your scientific question. Things to consider when selecting an assay system.... Make sure you plan ahead and consider these questions to save yourself a headache later on. Enhancer...that much faster? In reality, many biological questions are not conducive to selections and have to be... would be required to answer your experimental question. Now that you understand the difference between...

...address all your unanswered questions via text! (well, maybe not all of them. We still haven’t gotten to the...Center at CalTech? If so, you may have submitted a question that didn’t get answered live - but he kindly ...our YouTube channel. And with no more ado, the questions!  AAV Prep and Technique Q1: Is there any way ...While we have not tried it, it might be worth investigating for both Q6 & Q7.For epithelial cells of the...to me a very high dosage. Any thoughts?  A: The question refers to this paper. AAV can be safely concentrated...our participants for their time and excellent questions.    Additional Resources on the Addgene Blog ...

...be a good statistic to compare between samples. In cases where there is no clear distinction between the... colors. Here are some good practice tips on adjusting voltage: During data acquisition, make sure that... Negative and positive samples are clearly distinguishable as two separate populations. Created with biorender.com...these populations may overlap slightly, or the distinct populations may not be as obvious in a dot plot...that shifts to the left. When using MFI, you can still continue gating populations of interest with normal... red (bottom) peak overlaps with the baseline suggesting this sample has a comparable expression to the...

... complex consisting of multiple distinct effector domains that work together synergistically, modeled ...and control genes, but according to the Broad Institute's Silvana Konermann, the first generation of CRISPR-Cas9...them with the CRISPR system was a large one.  The question for her then was this: Can you engineer CRISPR-Cas9...just such a system, which they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an engineered...also available at Addgene, enabling systematic investigation of the cellular consequences of repressing or...

... activator-like effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers showed they ...binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding domain is modifiable ... into any cell type relevant to the disease in question – cardiomyocytes, adipocytes or neurons, for instance...Addgene: Tell me about the history. How had investigators done this kind of work before? Musunuru: In ...Unfortunately, it is time consuming and difficult for investigators without a lot of expertise to create zinc finger...and puts it in the hands of pretty much all investigators. Our kit makes it extremely simple to design...Musunuru: The CRISPR/Cas9 system is easier to use as testified by the fact that the TALEN kit includes 834 plasmids...

...experiments for every antibody that you receive; citing existing validation data can be acceptable. However, it...adequate validation. If you are not convinced by the existing data, you may need to roll up your sleeves and...yourself.  Goal of Antibody Validation  As you assess existing data or plan your own validation experiments, ...applications. Here, I want to highlight the general suggestions given by the International Working Group on Antibody...you are trying to show that your antibody can distinguish between two very similar proteins. Additionally...of your target across samples - it is hard to distinguish between specific, invariable expression and non-specific...laboratories. Additionally, it can be difficult to distinguish between proteins bound by the antibody vs proteins...

...expression, low levels of spurious expression were still observed in many systems. So what is the cause of...performs in your system. This includes a thorough testing of both the serotype and promoter with in vivo ...transduces your cells of interest. We recommend testing several different serotypes to determine which ...the serotype Since each serotype also exhibits distinct patterns of spread from the injection site it ...to transduce your cells of interest. Using a constitutively expressing vector for this step may save time...in order to minimize off-target expression while still achieving adequate expression in your cells of interest...recombination rate (0.1% and 0.13% respectively) still required a 200-300 fold dilution to minimize off-target...

...scientists frequently use growth factors or serum to stimulate a pathway of interest, but there can be a lot ...domains on either side of the active site of a constitutively active MEK1. This photoswitchable MEK1 is called... a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the ability... fluorescence can be found in the nucleus. For testing kinase inhibitors, psMEK1 and ERK KTR-mRuby2 expressing... KTR-mRuby2 system has several advantages over existing methods. First, it avoids activating other signaling...occurs when serum or growth factors are used as a stimulus. This system also allows precise modulating of... maintenance of phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative...

...them a quick scan for interesting key words and download what seems interesting or relevant. Harness the...though you may need to be on a network with an institutional subscription), archived on PubMed Central, or...information to allow you to understand the research question that will be addressed. In primary research papers...Is the data of sufficient quality (error bars, statistics, clarity in images etc)? Did they perform all... you prefer, printing and hand-writing notes is still possible, but not feasible for every paper you will...search. Writing this tutorial has shown me that I’m still in the dark ages with respect to my organization...

...Feel free to add other suggestions in the comments or email me with suggestions.  Most coaches have websites...seriously. As I make my rounds speaking at research institutions and at scientific conferences, I see that few...will be a clear communicator and make direct suggestions for improvement and change. Work with the coach...making this transition). Another coaching group is Still Point Coaching. One nice thing about a group is ...coaches I have mentioned. I have seen a lot of fantastic online content for PhDs and postdocs posted by...Coach De La Professional but the website looks interesting and it is in English and Spanish. She is based...

...patients have achieved persistent immunological reconstitution with a normally functioning T cell repertoire...variety of vector delivery technologies have been investigated including lentiviruses, adenovirus, and inorganic...? There are a variety of key properties that distinguish AAVs as gene delivery tools: Unlike most naturally...repair via homology directed repair (HDR). This characteristic makes AAV vectors especially amenable to correcting...but not limited to hemophilia B, LPL deficiency, Cystic fibrosis, Muscular Dystrophy, Parkinson’s disease...overcome key challenges to their efficacy:  Pre-existing immunity: Many individuals already have wt AAV...high as 70% among the human population. These pre-existing antibodies can interfere with virus-cell interactions...

...technological advances from exacerbating existing inequalities and injustices. The Personal Genetics Education...organizations, libraries, youth groups, and faith institutions. The conversations often move quickly into sensitive...sensitive topics and uncomfortable places, from sticky ethical dilemmas to dark episodes in the history...hands on deck” to take care of the organizational logistics. Setting yourself on the path to science policy...being very focused on narrowly-defined research questions in our field of interest. We often communicate... on public lectures, science cafés or science festivals, and getting experience in either giving or running...

...neuroscience, this architecture is frequently still an open question. The connections between neurons are what...addressed the question of how newly generated neurons integrate into the complex pre-existing circuit of ...a major part of the answer to many biological questions about the brain. To address this, molecular tools...continue to contribute answers to the complex questions of how the brain operates. References 1. Callaway...Tsiang H, Ceccaldi PE. Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: ...

...Planarians are easy to buy, cultivate, and have interesting phenotypes to study. In addition, the Sánchez...are 11 different genes cloned into plasmids to investigate for each of these planarian species. These plasmids...wildtype locomotion.   Create your own plasmids to investigate other genes in planarians! For those who are ... performing their own molecular cloning and investigating genes not in this collection, they may clone...stowers.org and if you make new plasmids for investigating different planarian genes, we encourage you ... other classrooms and researchers can easily investigate in planarians. References 1. Alice Accorsi, ...PMID: 9486653. 3. Newmark, Phillip A., et al. "Ingestion of bacterially expressed double-stranded RNA inhibits...

...CrispyCrunch may evolve over time, you can depend on it to stick to a few guiding principles that we've followed ...can see all the current code on GitHub, ask us questions about it, or send us a pull request for improving...genome browser, Ensembl, and others. It builds on existing popular web services, currently Crispor and Crispresso...designed outside CrispyCrunch, or you can input pre-existing guides for primer design. The ecosystem of CRISPR...deciding to build our own tool, we looked hard at existing tools. As has been written here on Addgene, there...HDR. Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide...hope it will do the same for you. Send us your questions or comments to [email protected]. We'...

...wealth of potential, and while many applications are still yet to be demonstrated (or invented), its behavior... into a variety of optogenetic tools, they’re optimistic that MagLOV can be similarly adaptable into future...-publishing directly on his website (and often posting updates on social media) and freely sharing code...results early and often, including works-in-progress still in need of further study and development like MagLOV...MagLOV. The results with MagLOV raise broader questions like “How and why do fluorescent proteins respond... speculate, they know they can’t answer these questions alone. They readily admit they don’t fully understand...more. If you’re interested in working on these questions or collaborating, the York Lab is eager to hear...

...antibody come into play? We’ll dive into those questions in this post as we explore… the life and times...and monoclonal antibodies. However, it was (and still is) infeasible to generate highly specific antibodies...target and basically impossible if you wanted to distinguish between endogenous and exogenous versions of ...significantly between human and the other species, suggesting that these antibodies recognize an epitope within...suitable pair for protein tagging. In two studies investigating mechanisms of endoplasmic reticulum localization...EQKLISEEDL) - larger than the substance P peptide, but still quite small. However, unlike the substance P tag...involve taking the Myc1-9E10 variable regions and sticking them onto different scaffolds. But over the years...

...Twitter - Tweet suggestions and tips with #NgAgo The Addgene Blog - Leave suggestions and tips in the ...November 18, 2016): Researchers from a variety of institutions recently reported their inability to recapitulate...nearly 400 times. Researchers have been excitedly testing NgAgo's genome editing applications. So, how does...successful, and the second section asked basic questions about the NgAgo protocols used by respondents ...epitope knockin was achieved by a few respondents suggesting that NgAgo can indeed be optimized for genome...PubMed PMID: 27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo...

...of a single microhomology sequence. For more mechanistic details on HDR and NHEJ, please see the linked... anneal incorrectly, MMEJ may also introduce, substitute, or delete nucleotides in addition to the expected...larger amount of material into the genome. Open questions and alternative systems The ready-made PITCh plasmids...cDNA constructs into this locus. One important question to ask is how the efficiency of PITCh-based genomic...genomic insertion would compare to CRISPR sticky-end insertion using the nuclease Cpf1. Since Cpf1 cuts...-independent knock-ins, but this possibility is still being explored. More broadly, Sakuma et al.’s use...nucleases and their applications. It will be interesting to see what new editing possibilities MMEJ can...

...2):a001107. PMC PMID: 20182618 . Do you have suggestions for other plasmids that should be added to this...