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...kinetics of the target ligand. Another important consideration is whether the affinity of an experimental protein-ligand...Proteolytic removal of the fusion protein could provide a more relevant kinetic analysis, but the trick...immobilization through biotin-avidin binding could provide a true measurement of high affinity (i.e. sub-nanomolar.... MBP has a similar function as GST in that it provides a method to express a foreign protein in bacteria...biotinylate the lysine residue in the AviTag. As a side note, we found that adding a TEV linker dramatically...that we couldn’t find an all-in-one plasmid that provided an easy way to biotinylate a fusion protein in...biotinylation and purification strategy outlined here provides a basis to determine binding kinetics using a ...

...transposon? The piggyBac™ transposon was originally identified in moths in 1983, but wasn’t successfully used... that have negative impacts on fitness. One consideration when making a transgenic cell line with the ...the piggyBac™ system, these challenges can be considerably reduced. The piggyBac™ transposon system can...donor-plasmid, you could use a single-stranded oligodeoxynucleotide (ssODN) as the donor [6]. The problem with... his specialized knowledge to help researchers identify the right technologies and strategies for their...sites is cytotoxic for proliferating cells and provides a simple tool for conditional cell ablation.,”...Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications...

...brilliant ideas on how to solve many therapeutic and pre-clinical problems! Who knows, perhaps the ideas spurred...students play, you can observe, answer questions, and guide them to productive strategies while noting any gaps...their knowledge. Practically speaking the format I decided on was to have two teams of 4 people face each ...with them. My impression was that they had clearer ideas about gene therapy in the following lessons. And...freely share your projects with us! This exchange of ideas will help improve life science and biomedical education...molecular biology educational resources Find protocol videos to teach basic lab techniques Plasmid Land: The...

...This post was contributed by Deborah Sweet, Vice President of Editorial at Cell Press. Almost everyone who...some point. Usually it comes up when a researcher decides on a new project and begins by trying to reproduce... various different versions of checklists and guidelines to help authors understand what they are expected...NIH) now look for data management plans and for evidence of sharing for grant renewal—but there is clearly...hiring, funding, and promotion processes, I’m confident that everyone’s enthusiasm for actually making...Sweet from Cell Press. Deborah Sweet is the Vice President of Editorial at Cell Press.  She trained originally...editorial work to focus on a broader role as Vice President. Her current responsibilities encompass overall...

...without changing them. Thus, it has been widely adapted for a wide variety of applications: base editing,...fusions localized to specific loci based on the guide RNA sequence. A year later, they developed CRISPRainbow...RFP) to the genomic loci of interest. Pairs of identical hairpins within one gRNA will result in a blue... chromosome-specific loci in the genome and the guides are relatively unstable.   Figure 1: CRISPRainbow...deliver CRISPR-Sirius into cells. They named this guide sgRNA-Sirius-8XMS2 which will recruit the HaloTag...Find plasmids for your research Read our CRISPR guide ...

...research from bench to bedside is, in addition to thorough ethical considerations, in making sure that ... our CRISPR guide Find CRISPR plasmids by function Check out our genome engineering guide ...scientists and the public together to build consensus guidelines, but there was never a specific timeline for ...shuffling our feet on necessary actions to review and decide on the future of genome editing technology. We ...Harley and Lexi, and a clutter ofoutdoors-only spiders, all of whom are named Charlotte, all of whom are...

...yoga classes. Get outdoors. Taking a walk or a bike ride is a great activity for both exercise and clearing... times of stress due to #COVIDー19. Receive and provide support while being safe. @GHP_HarvardChan — Shekhar... (individual development plan) from Science to identify skills to work on for the next steps in your career...networking events. Download Addgene’s Science Career guide to learn more about career paths and things you ... Everyone. Plan out new experiments carefully. Consider submitting a registered report, where peer review...is based on the methods, not the results. Also consider using this framework to design your future experiments...Technique. Co-work virtually. With co-working, the idea is not necessarily to chat, but to mimic your usual...

... took classes in biochemistry, microbiology, epidemiology, and bioanthropology. Studying human health ...doing in the lab and the interactions I was having outside of the “ivory tower.” I’d spend the afternoon lovingly...survived this long without downing any dihydrogen monoxide (H2O). Studying science communication as a PhD...quality of the study design or by their preconceived ideas about the controversial medical issues. Participants...beliefs even though all three texts were otherwise identical. Likewise, when participants read an abstract ...in a behaviour called the “biased evaluation of evidence,” in which people (conscious or unconsciously)..., looking for reasons to dismiss them. There’s evidence to show that people with greater levels of science...

...’ll be confident in what to expect. No system, including MTAs, is perfect, but it does provide consistency...1995: the UBMTA. Because biological materials are widely varied with many properties, having well-understood...Instead, we facilitate the signing of MTAs by the Provider and Recipient (in Addgene parlance, the depositor...plasmids are distributed under the UBMTA, which is widely used across academia. It’s our process that differs...to note that it’s the depositing institute that decides the terms of distribution for their plasmids. ...Webpage, help center articles, and our MTA YouTube video for more information. Happy transferring!    Many...

...Services - includes videos on general resume writing, as well as additional videos with emphasis for graduate...Relations Director for Curiosity Collider (hyperlink https://curiositycollider.org/) , which she co-founded, ...applying for the position?   Here are a few things to consider when you're preparing your resume for a non-academic...you only have two pages, you need to have a good idea of what should be highlighted on your resume, and...postings and look at the jobs you are interested in. Consider listing the skills you already have, and the skills...

...collection that grows on a daily basis, the lists below provide a snapshot of relevant resources and a good jumping...the gene you’re looking for on this list, please consider searching directly on our website. In-exhaustive... from a Set of Mitotic Chromosomes. Samwer M, Schneider MWG, Hoefler R, Schmalhorst PS, Jude JG, Zuber...DW Cell. 2017 Aug 24;170(5):956-972.e23 Screen identifies bromodomain protein ZMYND8 in chromatin recognition...receptor signaling. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR Cell. 1998 Nov 25. 95(5)...chromatin and the epigenome. If you have been considering some new techniques, the posts below may steer...

...Microscopy, University of Amsterdam. Before you decide which car you want to buy, it is worthwhile to ...tables with fluorescent protein properties and provides a quick way to compare fluorescent proteins, e.g... the protein of interest by a 2A self-cleaving peptide (Goedhart et al, 2010). An explanation of the assay...proteins (with the same reference protein) under identical conditions, a ranking of the practical brightness...practical brightness. Therefore, practical brightness provides a better picture of what can be expected in a ...theoretical brightness. Consequently, it makes sense to identify a couple of promising fluorescent proteins and...mimicking the future application. In figure 2 we provide an example of the comparison of the practical brightness...

...Viral Service. With their support we are able to provide faster access to a larger number of viral preps...Karpova’s lab from Janelia Research Campus have provided valuable insight into AAV production. Finally...advice and encouragement. Why virus? Why did we decide to take on this project? Addgene’s mission is to...plasmids in a ready-to-use viral format. Having identified this need in the community, we approached it ...control, generates  educational materials, and provides viral vector technical service (details about ...expertise in answering questions on virus and helping provide educational resources on this collection. With ... we could ship internationally and be ready to provide extensive logistics support for export and shipping...

...Encourage your close peers to come along for the ride, and consider applying to several opportunities. In an ...academic resources, we created STEAMid, for a nationwide database of  STEAM internships for high school...order to gain a competitive advantage this cycle, consider the advice below:  1. Eligibility check Scrutinize...and grades that serve as automated filters of consideration. If you are worried about any eligibility provision...to writing a solid personal statement will pay dividends. This is the only opportunity where you get to...ahead for success. Every student should use these guidelines consistently across a number of years as you ...

...subcellular localization peptide. By combining three different localization peptides with four different fluorescent...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...nanobody-GFP interaction). The modularity of the system provides researchers better flexibility and makes the approach...therapeutics to the CNS, and with reduced systemic side effects, by allowing the user to select for variants... those shown here, only AAV.CAP-B10 causes brain wide expression, with no liver expression, after retro-orbital...mixed-subclone system using mStrawberry, localization peptides, and reporters that drive the expression of the...

...the lab and you're sweating the small stuff? Identifying the Issue You can’t fix an experiment by simply... work. Draw your plate maps onto your plates, alongside your physical copy. If you’ve got messy handwriting...experiment than it is to rerun an experiment, so consider your prep an investment in accuracy.  Bench setup...you add them. Moving your reagents from your left side to your right as you use them can help you remember...to whom I am forever grateful, I started setting aside 3-5 minutes to sit quietly at my bench before the... to a protocol if you’re in a hurry, but let’s consider it this way: When you’re stressed or rushing, ...20Blotting.pdf  More resources on Addgene’s blog A Guide to Getting Started in Undergrad Research Early Career...

... AAV (rAAV) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant...biorender.com. ITR structure  WT ITRs are symmetrical 145-nucleotide sequences that flank the ends of the single stranded...Figure 2). Additionally, the ITR contains a 4-nucleotide Rep binding element (RBE) that serves as a binding...sequences. GC tails are often located immediately outside of the ITRs in the plasmid backbone and do not ...since analysis of individual reads allows for identification of non-dominant ITR sequences.  Restriction...Capsids More resource on Addgene.org Addgene's AVV guide AAV Viral Preps References  Chen, Y., Hu, S., Lee...Alexander, I. E., & Logan, G. J. (2019). A User’s Guide to the Inverted Terminal Repeats of Adeno-Associated...

...infectivity): The use of the VSV-G envelope provides the widest tropism or range of cells a virus can infect...The widespread use of γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted...into the 293T packaging cell line. This system provides the greatest flexibility to pseudotype γ-retrovirus...encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the packaging cell...terminal repeats; U3-R-U5 regions found on either side of a retroviral provirus (see below). Cloning capacity...Protocols: General Transfection The Addgene Retrovirus Guide  ...

...can be engineered to be cell type specific. Once inside the host cell, the virus unleashes its cargo – ...transduction efficiency and can effectively infect a wide range of cell types that are typically ‘difficult...viruses have a low immunogenicity which makes them ideal for applications with whole organisms. Finally, ...right approach for your experiment, you’ll need to decide exactly which type of virus to use. Types of viruses...genome into the host genome, setting up permanent residence. They primarily infect dividing cells, as their...signals which can traffic their viral particles inside the nucleus, without the need for envelope disassembly...clinical applications, viruses are true bench to bedside tools. Keep an eye out for our upcoming blogs on...

...modification of multiple genes is often necessary. Guide RNAs (gRNAs) are commonly packaged in 400-500 bp...terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit the number...cleaved precisely as predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified ...tRNA-gRNA-tRNA constructs. gRNAs with any 5’ leading nucleotide could be processed, a distinct advantage over...over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors indicate...frequency at the four loci, indicating that Cas9 can be guided to at least 8 distinct loci. They also examined...this publication at Addgene Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing...