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... with DNA sequencing troubleshooting and analysis 1. You can use any of the following programs to view...of interest. 5. Use a silica spin column for purification of the samples you send for DNA sequencing If...requires you to perform your own Big Dye PCR amplification reaction (as opposed to using the all inclusive...

...Histochemistry & Cytochemistry, vol. 59, no. 1, SAGE Publications, Jan. 2011, pp. 6–12. Crossref, doi:10.1369...compare the experimental group.     Figure 1: A, Most biological experiments will benefit from ...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding...blot and look for the molecular weight shifts indicative of tau phosphorylation. By including the positive...

...science’s place in education, government, and civilization in general (1). Science and politics? Science...consequences of a march. Three of these risks include: 1) Lost trust Perhaps the greatest risk is that the ...sister locations on April 22, 2017, scientists and non-scientists alike will march to advocate for science...already come to pass. It may only be by advocating for science education and literacy that this changes.  2...marching for. I will be marching to advocate for science education and literacy as I believe people that...communities, microbial interactions, and science education.  Disclaimer: The views presented in this article...process begins with a testable hypothesis - an educated guess about how some part of the world functions...

...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...sensors One of the latest additions to Addgene’s catalog is a new class of sensors for neuropeptides and...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...PDGFR.codonopt    Aggarwal A, et al. Glutamate indicators with improved activation kinetics and localization...have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022) optimized...

...detects the light scattered by that cell at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values ...colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered forward at a low angle... you with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements...exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably...types of scattering that occur when a cell is pulsed by the laser. Within each type of scattering, you also...to keep it short though, promise! FSC and SSC: scattering To digitally extract your cells based on their...passes through the laser beam and causes it to scatter (FSC/SSC), the detected photons can be represented...

...together along with the Gibson assembly master mix for 1 hour, and then transform into competent cells. The...you should consider giving it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease...

...example, if there are 10 putative off-target locations you can 1) determine if editing at those sites would...downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case, the ...“Off-target effects” a vague catch-all term for unintended consequences in an experiment. For CRISPR ...sequencing. While this may be unnecessary for many applications, it is the only comprehensive method available...binding by Cas does not always correlate with catalytic activity at those sites, especially when DNA mismatches...For a candidate site-specific approach, mRNA quantification or western blot of putative off-targets would...you’ve tried everything listed here. For some applications, this is ok, but it still must be controlled...

...out these SciComm blog posts. References 1. Bourdeau, Raymond W., et al. "Acoustic reporter genes for... science communication and outreach - if you'd like to learn more about Science communication check out...microscopes and give a similar optical quality (magnification of 140X and 2 micron resolution). I have worked...creative approaches to visualize bacteria in an educational setting. The Symposium on ‘Creative Approaches...from hand-drawn comics (Dr. Sanja Saftic) to educational gifs created by the Amoeba Sisters. Here’s one...Bacterial Genome Engineering Learn about Science Communication using Twitter  Resources on Addgene.org Check...

... remains fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in...follow him on twitter: @joachimgoedhart.   References 1. Zacharias, David A., et al. "Partitioning of lipid-modified... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...an additional protein module that can report on location. This approach enables the study of proteins in...fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...case when proteins undergo post-translational modification with fatty acyl chains. For instance, myristoylation...C-terminal consensus sequence (-CaaX). Both modifications are disrupted when GFP is fused to these sequences...

...may contribute to more in the future. Figure 1: A timeline of CRISPR base editing advances that led...Lander, E. S. (2016). The Heroes of CRISPR. Cell, 164(1), 18–28. https://doi.org/10.1016/j.cell.2015.12.041...Advancing from this invention to KJ’s clinical application in under a decade is an astounding feat of public...effectiveness varied depending on the chromosomal location, sequence context, and cell type. Multiple labs...of identifying a successful editor. For this application, ABE8e turned out to be most effective. Targeting...editors with increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https:...

...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...virtually. 6. Quantifying DNA? Here are Five DNA Quantification Methods to Consider by Jennifer Tsang The...The nanodrop vs. spec debate dominates DNA quantification discussions, but what about the other methods... with plasmids deposited at Addgene! 2. DNA Purification Without a Kit by Beth Kenkel To complement ...

... two years. References 1. Martz, Colin A., et al. "Systematic identification of signaling pathways with... of 17 signaling pathways that are frequently implicated in cancer cell proliferation, survival, differentiation... make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood and his...

...infections, cancer, and other diseases. References 1. Harrington, Lucas B., et al. "Programmed DNA destruction...of dsDNA and RNA in trans. Fluorescent signal indicates the presence of this viral sequence in the assay...

...regulation of endogenous RNA. J Biol Eng. 2014 Mar 1;8(1):7. doi: 10.1186/1754-1611-8-7. PubMed.  Related Resources...says the new tool might even find therapeutic application in addition to their use in basic biology, for...

...to the interview with Science in the News Chapter 1: Introduction - Vini and Amy tell us a little about... to be crazy complicated! Listen to the full podcast for more great science communication tips or listen...tons of ways you can get involved in science communication. In this second half of our conversation with...many things you can do start your own science communication student group and get more involved with your...is a graduate student group with the goal of communicating science to the general public and training the... the next generation of science communicators. In this chapter we learn about some of their events - science... peer-to-peer feedback in effective science communication. Chapter 7: The Goal of the SITN Podcast - Vini...

...deletions, and all possible base-to-base changes.   1. Plasmids 101: Transformation, Transduction, Bacterial...resources can now be found in one place: the Educational Resources page. So let’s get to it. Here’s the...Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins by Cary Valley,...protein size and shape, linker sequences, and fusion location, there are many choices to make. 2. Prime Editing...Resources at Addgene Browse the repository Find educational resources at Addgene Search for plasmids at Addgene...

...Zhang Lab's Nature Protocols Publication describing them here. References 1.Ran, F. Ann, et al. "Genome...

...2013 edition. 10 new developments in open science 1)      In February, the Office of Science and Technology...support for open access to peer-reviewed journal publications, although many don’t necessarily publish in ...

...P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in promoter...lentiviral transduction. Lysine-specific Demethylase 1 (LSD1) Tatjana Sauka-Spengler's lab has deposited ...epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting...DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic... the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified...Preps for Epigenetic Modification Why use epigenetic modifiers? Epigenetic modification is certainly not ...Nature Communications 8: . https://doi.org/10.1038/ncomms16026 Find plasmids from this publication at Addgene...

...to western blotting  In a western, proteins are: (1) separated by size, (2) transferred to a membrane,...  (3) detected using antibodies.   Figure 1: Overview of the Western blot process. The sample,...post-translational modifications, quantify protein levels relative to standards, detect the cellular location of proteins...pull-down assays.  Westerns are divided into two categories, native and denaturing. In a native western, ...matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper. Pretty much...search, first pay attention to the validated applications listed on the vendor’s website and choose an...western and vice versa.  In addition to validated applications, pay close attention to the controls listed ...