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...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...

...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...

...with serial dilutions of the lentiviral vector, harvesting cells several days later, and isolating genomic...

...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...

...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...

...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...

... 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...

...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...

...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...

...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...

...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'...

...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...

...260 nm, and 280 nm. Nucleic acids exhibit a characteristic absorption spectra across this range of wavelengths...absorption at 260 nm (A260). Figure 1: Characteristic absorption spectra for pure DNA and likely ...phenol, or guanidine salts, exhibit their own characteristic absorbance spectra, which you can detect by...systems. Peak absorption occurs below 230 nm, with a distinct secondary peak around 280 nm thanks to aromatic... even samples with “good” A260/280 ratios could still have quite a bit of protein present.  Purification... RNA and DNA absorb maximally at 260 nm, so distinguishing between the two can be difficult by spectrophotometry... concentrations. However, such systems are no substitute for sound techniques, routine machine maintenance...

...interested in sharing your science, one of the big questions you'll be faced with is "how?" How are you going...to Rule Them All, consider the following four questions to figure out which method(s) work best for you...You never know what's going to work. After all, a stick-figure internet comic about math probably didn't...Selecting the method Once you’ve answered the above questions, you can create your short list of options and...low data requirements.  No visuals. Variety of hosting platforms can splinter audience. Primarily digital...difficult to convey an entire story or context via still images. Some things cannot be photographed or need...print distribution.  Static. Often requires both artistic and scientific training. Cartoons may be read ...

...developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular interactions...instance, the DBD of the fusion protein shown is still able to bind to the upstream activation sequence... is an example of the former. In this system, histidine-auxotrophic host strains are used to select for...expression of the HIS3 gene, which is required for histidine manufacture and therefore growth on his-deficient...split-luciferase system, intein-mediated splicing reconstitutes functional luciferase only when fusions of the...high number of false negatives in Y2H screens. Testing your reporter system with a pair of proteins that...Y.C., Rajagopala S.V., Stellberger T., Uetz P.  Exhaustive Benchmarking of the yeast two-hybrid system. ...

...AnltWfKuQg pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020 Contest 2: Deck ...temperatures, our first of the season snow flurries, dusting off the winter coats and hats, and our annual Deck...lab space or your work from home space in a fun, festive holiday or winter theme following the rules outlined... pages. Make your WFH space feel a little more festive this holiday season! It is that time of the year... fun, be creative, and enjoy the end of year festivities - however you celebrate! And if you want to see...

...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid protein...fluorescent signal at baseline voltage), the investigators found that administering these AAVs with a dilute...has developed REVeRT, for gene delivery using reconstitution via mRNA trans-splicing. By splitting a large...their mRNAs, the functional gene product is reconstituted within the target cells (Figure 6).   ...Illustration of split fluorophore assay to test reconstitution via mRNA trans-splicing. A cis-splicing vector... The authors used REVeRT to functionally reconstituting coding sequences for a range of applications...introduction of extraneous genetic material to reconstitute the gene of interest, reducing the immunogenicity...

...following unanswered questions about CRISPR screen design. Unanswered questions about genome-wide CRISPR...exist to control for these faults. However, it’s still unclear if similar pitfalls exist for CRISPR screens... rates than their non-targeting counterparts, suggesting that safe-targeting guides are more toxic than...closely mirrors that of gene-targeting guides, suggesting that safe-targeting guides better control for...also reproduced several previously observed characteristics that influence gRNA off-target activity: 1)...presents a strategy to control for these effects. By testing thousands of guides for off-target activity, Morgens...

...experiment-based investigation of a hypothesis: proper controls! For every experiment, an investigator needs a ...the effect of the independent variable you are testing. Importantly, they demonstrate the functionality...associated with the independent variable you are testing and not some other unintended factor. Control plasmids...control plasmids would address and answer these questions. Types of control plasmids Part of planning your.... An internal control may be a plasmid that constitutively expresses a reporter protein (e.g., GFP or ...tenet of responsible scientific inquiry and investigation. References: 1. The problem of pseudoreplication...

...AnltWfKuQg pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020   Deck the Lab: ...Edition Winners In a tie for first place, the Vestibular Lab decorated not just their lab, but their entire... entire floor, and put together a festive video to showcase it all!           View this post on...TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated with dozens of individual virus snowflakes...cookies! 🤗 🤗 🤗 We hope everyone has a safe and festive Holiday season. 🙌 🥂 🎄 🎉 😷 Remember wear a ...

...genome-editing tool but it could be a fantastic add-on to a diagnostic toolkit, called DETECTR, which has...iceberg as it has been predicted that many others are still unknown. Identifying Cas14 variants In this quest... from the Department of Energy’s Joint Genome Institute for uncharacterized genes that were proximal to...presence of a predicted RuvC nuclease domain, characteristic of CRISPR-Cas enzymes. In contrast to other...middle of the gRNA to be activated. Cas14 in diagnostics By cutting ssDNA but not dsDNA Cas14 is not a...

...are packaged, the physical size of the genomes is still ~4.7kb. But despite this, larger functional transgene...long between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al...site donor and acceptor sequences are used to reconstitute the two pieces of the transgene.The AAV encoding...pre-mRNA of the two halves of the transgene will reconstitute the full-length transgene.  Drawbacks:  Trans-splicing...half of the transgene. The full length gene is reconstituted by either the overlapping or the trans-splicing... template’s integration so that the site is reconstituted after integration; and a sequence of stuffer...since there are two ways for a transgene to be reconstituted, there is currently no one ideal split vector...

...cyanobacterium Synechocystis sp. PCC 6803." Biology open (2016): bio-017129. Hsia, Justin, et al. "Modular...you’re an academic researcher at a nonprofit institution, you can order plasmids covering a wide range...how they’ve been used to date . If you have any questions, please write to us in the comments section.  ...Alexandrov lab at the University of Queensland’s Institute for Molecular Bioscience has provided a solution...library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free expression...The gene set was carefully chosen to perform statistically relevant benchmarking of cell-free expression...

...phosphatase treatment, substituting cytidine with 5-methylcytidine, and/or substituting uridine with pseudouridine...behind at the site of excision, this approach is still not considered safe for therapeutic applications...screening cells for the presence of transgenes is still an important control. Additionally, Sendai virus...The typical drawbacks of using transfection will still exist: transfection efficiency is cell-type dependent...by dilution with each cell division, but it can still take several passages for the minicircle to be completely... that ⅔ of subclones generated from EBNA1 iPSCs still had episome DNA present. DNA-Free Methods RNA...reprogramming proteins for 8 hours a week for 6 weeks, and still required further culturing to generate iPSC colonies...

...Zika Virus As always, if you have any questions or suggestions for us, feel free to comment below. Happy...in DNA. The first, contributed by guest blogger Kristian Laursen from Cornell University, is a bit old ...discussed further in this informative post. Interestingly, this technology may make some uses of Site ...Honorable mentions Of course we’ve had many other fantastic posts throughout the month including a first of... Software Engineer Kris Shamloo wrote a very interesting post about how we develop our in house lab management...

...negative FRET controls or calibration standards consisting of donor and acceptor proteins fused with linkers...