...recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method. Read...on to learn how to use it in your research! The starting point: Ligation-independent cloning Ligation-independent...nucleotide, T4 will switch to polymerizing and then stall due to the absence of the other dNTPs. Once the ...of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process, a gene of interest is ... SLIC can’t be used if the overhangs would have stable ssDNA secondary structure — for example, the stem-loop...enzymes required for Gibson assembly. SLIC is a standardized method for multi-fragment DNA assembly, and ...

...your actual samples. They may also be used as a starting point for compensation (Fig. 1B-C). However, it... The single color controls should be used as a starting point, to preserve your sample, and you should... compensation (C) with APC-Cy7.   FMOs FMO stands for fluorescence minus one. It is used as a negative... samples, combine it into one well or tube, and stain it with every marker you are using in the experiment...and be in the same fluorophore (e.g., if you are staining for anti-mouse Ly6C in APC with the IgG2c subclass...a combined subset of your experimental samples, stain it with every marker you are using in the experiment...

.... To get started, you will first need a primer that complements your plasmid sequence. Start with a backbone-specific...plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone that contains the plasmid... lost a specific gene survive (ex: antibiotic resistance marker). In a previous blog post, we covered ...plasmid backbone, as it often relies on antibiotic resistance. But what about identifying clones that contain...Lynne Postovit’s lab. It contains an ampicillin resistance marker in the backbone. A selection on agar containing...

...don’t have a background in statistics and programming, you’d be wise to start working on these skills. ...techniques from across different disciplines to better understand neurobiology and human cognition. Developing ...interaction between neuroscience and other fields. For instance I met one researcher studying how the microbiome... apply your knowledge of metabolism to better understand how aberrations in brain biology lead to altered...always suggest a plasmid to us, request that we start making the AAV, or help us produce new educational...

...mechanism by which bacteria have gained antibiotic resistance and virulence. Scientists have studied how HGT... Guillemin, 2006) or the presence of specific substances (Meibom et al., 2005). Though not all bacteria... replication-incompetent phage particles with assistance from a ‘helper’ phage prior to transduction. ...expressed but not integrated into the genome and stable cell lines, where foreign DNA is incorporated into...can infer genetic advantages such as antibiotic resistance. Bacterial conjugation as explained by... genetic material can pass through. If you are starting any molecular biology experiment check out Addgene...

...why the results you've measured should change established theory. So, what can you do to ensure that your...increasingly available. But think of this as a jumpstart or a refresher for your skillset rather than a... asks a question about something they did not understand about your presentation, question whether other...scientists need to communicate with non-scientists, to establish broader impacts for their work in education and...our attendees there online with #comscicon18 and stay tuned to our website and social channels to apply...organizations that practice science communication or start your own! When you do, let us know!  To get in touch...

...practices (QRPs) can quickly lead us to find statistically significant results that are not robust. Sampling...Preregistration is the process of creating a time-stamped research plan in a public repository before conducting... is typically done right before data collection starts, and should include a specific plan for how data...each hypothesis will be tested with a specific statistical test.  Preregistration makes clear the distinction...to present that work using hypothesis testing statistical tools that generate a p-value.  As an example...sampling noise with registered reports, the gold standard for testing hypotheses Finally, Registered Reports...

...rigorous QC standards are met, we are taking a unique hybrid approach compared to our standard pipeline....samples, the rest will be put into a status we call “Archived.” This status allows us to store plasmids and...pipeline. For the first ~50 plasmids we perform our standard full plasmid sequencing by NGS. Addgene’s QC scientists...contain all expected features and meet our QC standards. If any concerns are identified, we can quickly...

...Cas12a for multiplexed gene perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system...anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and Hiroshi Kimura’s labs have recently published...onto a well-folded scFv scaffold to improve its stability in cells, and use it in a variety of applications...complementarity determining region of an anti-FLAG scFv and a stable scFv scaffold. (b) Schematic of anti-FLAG-FB-GFP...2021. doi: 10.1038/s41587-020-0600-6. The Penn State Protein Ladder  by: Rachel Leeson This simple, inexpensive...expression and purification. Figure 3: The Penn State Ladder run on SDS-Page gels. A: individually expressed...

... attention on transcription factors, starting with an established method called ChIP-seq. ChIP-seq relies...explains it, a major goal in his laboratory is to understand the function of the non-coding portion of the...transcription factor. It also gives cells antibiotic resistance to neomycin so that you can select the population...We’ve done comparisons of our new system versus standard ChIP-seq for transcription factors known to have...available to anyone for a modest cost without having to start all over or write to us to get them. Even though...

...Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use information...genes that, when turned on, make melanoma cells resistant to treatment with a BRAF inhibitor. (The test ...system and contains 10 sgRNAs for each transcription start site in 15,977 human genes. The transcriptional ...library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman and his...manipulated at one time. Weissman’s group has made standard protocols available and they are working to further...gene, go to http://sam.genome-engineering.org/. Crystal structure of Cas9 in complex with guide RNA and...

...elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one of the key...neuroscience research where optogenetics will be a standard tool used in nearly any neuroscience project or...Intramural Research Program (NIH-NIDA IRP), with the stated goal of learning behavioral optogenetics in order...been published at this point.  In brief, some standard opsins (see table above) include Channelrhodopsin2...silence neurons than HR. Delivering your opsin  The standard opsin delivery method is local injection of an...considerations for your optogenetics experiments. Stay tuned!  Many thanks to our guest blogger Derek ...

...incredibly useful transferable skill. Being able to stand up in front of an audience and convey your ideas...you need it, rather than trying to push through a state of panic. Now, if I feel myself getting too nervous...present a paper to our classmates so they could understand it. When my mind went blank, my options were ...sense. As tempting as option two was in my panicked state, only option one would let me accomplish my goal... me.) They happen to everyone! Everybody makes mistakes, encounters technical difficulties, and runs into...you or not going perfectly, you truly are a rock star.  The best thing about public speaking as a scientist...

...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...into the system, it will bind and stabilize the DD, resulting in sustained protein levels. Looking for a ...turning on expression of a new protein is a cinch. Stably introducing the coding region of a gene (cDNA) ...mechanism. When a protein is tagged with a DD, it is unstable and quickly degraded. However, if the relevant...

...Schiebinger, PhD, Hinds Professor of History of Science, Stanford University. Sex and gender are critical components...Commission, the US National Science Foundation, and Stanford University—that harnesses the creative power of...Gendered Innovations. This project: 1) develops state-of-the-art methods of sex and gender analysis and...sexes come from, one clue or potential place to start research is by considering sex disparities in disease...important point, however, is that cost at this early stage of research is minimal compared to that of human...trials. And, as Janine Clayton points out, late-stage clinical studies that draw from a body of research...

...blots, for instance, almost always use the indirect approach, while flow cytometry’s standard approach ...targeting the Fc region of an antibody, the so-called ‘constant’ region which is, quite usefully, both conserved...cannot recommend it except in the most dire of circumstances.) Multiplex assays Mo’ proteins, mo’ problems... antibodies used are mouse IgG antibodies, for instance, an anti-mouse secondary will bind to all the ...flexibility in assay development. Researchers who understand how and why to use secondary antibodies will ...

...each ligation reaction uses a pool of gRNAs as starting material, the ligation products are a diverse ... al., 2014) and microRNAs (Wong et al., 2015). Starting your CombiGEM-CRISPR experiments The first thing...efforts made by various research teams to build and constantly improve gRNA libraries, excellent resources with... Wong et al 2015. Alan Wong is currently an Assistant Professor at the School of Biomedical Sciences... applying new technologies to interrogate and understand complex biological systems. He can be contacted...Timothy K. Lu. "Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial...

...to discuss their visions for a successful and sustainable biomedical research sector at our first Future...at this event added to a growing dialogue on the state of a system that, despite all of its successes, ...years of earning their PhDs (3), showing that the ‘standard’ pathway into academia may in fact be just another...Andrew Young School of Policy Studies at Georgia State University, is an expert on the academic labour ...postdocs. Among other involvements, he helped to establish grants through the Sloan foundation for postdoc-related...uses synthetic biology to create tools to better understand human disease.  Outside the lab, he is interested...

... addition to sequencing plasmids, we have also started using NGS to check the quality of other services...quality data! Addgene's new in-house NGS process To start, the process is only made possible by the use of...quickly create Illumina sequencing libraries. From start to finish the library preparation process only takes... individually assembles the reads into a single FASTA sequence that our QC scientists can easily analyze...performed, optimized as needed, and cleaned up using a standard PCR cleanup kit to remove any template DNA and...as part of our OpenBio Repository sometime soon. Stay posted!     References Guerin K, Rego M, Bourges...

...-fold more photostable and as bright to mVenus, the parent YFP, and is the most photostable YFP to date... were exposed to blue light. Cre-Vvd showed substantially improved activity as compared to other recombinase...addition, Casɸ has a minimal PAM requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where....ncbi.nlm.nih.gov/32675376/ mGold, the most photostable yellow fluorescent protein to date By Justin ...contribute to experiments that require high photostability, such as time-lapse imaging or with high light...