...certainly institute better experimental design practices including talking to statisticians when necessary...recommend watching the whole livestream for a more holistic understand of reproducibility issues and their...18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end... to future work cost both time and money with estimates reaching in the billions of dollars (Freedman ...ll see in Steve Almo’s talk about work at the Institute for Protein Innovation (IPI) (49:45 - 1:18:45)...apply these findings in their own work ultimately wasting both time and money. The obvious solution to this...experiments with too few replicates leading to suspect statistical significance (you can read more about this problem...

... control, you will be able to confirm that the testing protocols elicited the expected response and have...the precision and accuracy and therefore the statistical power of an experiment. Technical replicates ...meaningful results.  Biological replicates are distinct samples that are treated identically, used to ...knockout.  If knockout samples are unavailable, try substituting with knockdown approaches using siRNA or shRNA... block positively charged groups with generally sticky proteins such as bovine serum albumin. Secondary...either by modifying the blocking conditions or by substituting for a more specific secondary antibody. The ... a mouse IgG2a isotype primary antibody but you still observe a signal from your anti-Mouse IgG2a secondary...

...for testing and optimizing your serotypes, titer, and promoter. Note that several steps in testing are...you know that, you’re ready to start selecting, testing, and optimizing three key variables of an AAV vector...transduce your cells. In fact, we usually recommend testing more than one.  Most AAV serotypes have broad tropism...harm, your experimental goals when designing and testing your AAV.  Figure 2: Comparison of AAV ...cells express your gene. Off-target expression can still occur (Fig. 3) - for example, some neurons can transiently...selected potential serotypes and promoters to begin testing, you’ll next need to decide how much virus to put...Konishi, H. (2014). A comparative analysis of constitutive promoters located in adeno-associated viral ...

...so let’s go over the details step-by-step. 1. Digestion Set up restriction digests for your insert (or...until completion. The time required for complete digestion varies for different enzymes. Many companies now...have compatible overhangs or no overhangs after digestion, you will need to use a phosphatase to prevent...vector, unfortunately you can’t just throw the digestion mixtures together. You need to isolate your insert...compatible overhangs generated by restriction digestion to anneal to one another and form a complete, ...colonies are a result of uncut empty plasmid, you will still have colonies when you do not add ligase. If the...the plasmid After purifying the DNA, conduct a diagnostic restriction digest of 100-300ng of your purified...

...human reproduction, the relevant document for the question of “how do we move forward” is the June 2012 report...report is that when it became clear that this assistive reproductive technique was feasible, three separate...funded a center for mitochondrial research to investigate the scientific possibility, safety, and efficacy...efficacy of the mtDNA transfer options being investigated; A UK regulatory body tasked two separate non-governmental...remembered kindly. However, despite many headlines questioning if He’s experiments prove otherwise, the scientific...adequately safe or effective for use in humans. The question we are left with is not “what is the translational...all of whom are named Charlotte, all of whom are still working on theirpenmanship. Additional resources...

... restriction digest analysis. Diagnostic restriction digest Diagnostic digests can be used to confirm...be done right in your lab in less than a day. Diagnostic restriction digests are comprised of 2 separate...easier for you to obtain a useful and informative diagnostic restriction digest. For your digest: Try choosing...Consider buffer and temperature compatibility when digesting with more than one enzyme. Consult the manufacturer...The dyes also run at predicted sizes so you can estimate how far down the gel your bands have traveled ... diagnostic digest...

...opened with a very interesting presentation by Professor Guido Kroemer from the Institut Gustave Roussy. ...Research tackling questions in the fields of Cancer, Inflammation and Immunity, as well as various combinations...Findings in previous animal studies suggested that fasting has a positive effect on cancer treatment (Ref ...Immunosurveillance and Defense With increasingly sophisticated visualization techniques it is possible to follow...Professor Caetano Reis e Sousa from the Francis Crick Institute concluded “Aspirin promotes autophagy, inhibits...26343581. PubMed Central PMCID: PMC4597191. 5. Eyquem, Justin, et al. "Targeting a CAR to the TRAC locus with...

...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 ...

...appropriate ligand to activate the G proteins. It is estimated that there are approximately 950 different human...signaling cascades as a response to the extracellular stimuli. Ligand dissociation from the GPCR allows for binding...PRESTO-TANGO: The ligands that activate many GPCRs are still unknown. To identify some of these unknown ligands...2015). PRESTO-TANGO uses a modified Tango beta-arrestin recruitment assay to measure receptor activation...this assay, binding of an agonist recruits a beta-arrestin-TEV protease fusion to a receptor-transcription...adrenoceptor. GPCRs in chemogenetics  While there’s still a lot to learn about GPCR signaling, researchers...activated upon binding of CNO thus showing no constitutive activity. There are an array of DREADDs that...

...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...

...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...

... 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...

...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...