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...though. Typically, you will identify a ‘scientific mentor’: someone who provides career advice and offers... and identify any gaps or questions you have, then bring them to your mentor. Pro tip! Consider physically... a quality mentor, and rightfully so! They can provide you with high-level career advice, open doors for...Training follow up After your training session (ideally in the next few days or weeks), look over your ...protocol, or maybe you would like to take pictures/videos of a machine set up while your mentor is demonstrating..., your bench mentor’s training time is time set aside just for you – so do your best to show up as your...

...When choosing beads, there are several factors to consider. Agarose beads are sponge-like and vary in shape...shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated ...to the capture antibody and is immobilized. The ideal antibody concentration to use will vary between ...antibody is functioning as expected. In order to identify any non-specific interactions, perform parallel... is used to isolate the protein of interest. The wide availability and high specificity of anti-epitope...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect...routinely use the chromatin IP (ChIP) technique to identify DNA binding proteins involved in histone modification...

... live it out, but ideally without asking too many (annoying) questions. Identifying and solving problems...was aware that such roles existed, but did not consider them for myself. I started picturing myself in...Other Addgenies and Their Careers in Our Career Videos What's required for a role in technical support...any incoming requests or problems - there is considerable diversity in the types of questions customers...consuming and/or less technically challenging. Alongside there is a second-line team that takes on cases...problem. It may not always be straightforward to identify those crucial details because there are just so...Visit the Addgene Careers Page Check Out Our Career Videos ...

...recombinant Adeno-associated virus (AAV) has been widely used to deliver genetic tools to the brain because...neuronal circuits. Epitope tags are short antigenic peptide sequences, attached to a protein of interest (POI...simian virus 5-derived epitope (V5), the synthetic peptide FLAG, the synthetic streptavidin-binding strep-...both (Viswanathan, 2015). For Looger’s team, an ideal probe should combine the solubility, cell tolerance...imaging experiments, the second color is usually provided by red fluorescent proteins such as tdTomato or...smFPs can label fine neuronal structures with high fidelity and at lower concentrations than GFP. smFPs enable...N-cadherin in neurons and show that smFPs fusions provided better labels than fusions to 3 or more HA tags...

... will outline a simple method for selecting the guide RNA, validating its efficacy in vitro, and using...d like to manipulate in mind. Now it’s time to consider where you would like to attack this gene. Should... When the target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR... It can take roughly 3 months from the day you decide to do a gene modification experiment in mice to...have your F0 generation (barring life’s little accidents and stuff ups). This is a huge improvement on ...production of mouse lines that could be used to study a wide variety of phenomena, it’s possible that we’ll learn...of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome research 24.1...

... in lower lentiviral titers. Another factor to consider is the age of your culture post thaw. In general...lower titer virus. In such cases you may want to consider concentrating your viral prep. Storage can also.... For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. ... start to a successful infection, one point to consider is how the virus is being titered. Most titering... that routinely produce lentivirus may want to consider testing a variety of cation and serum sources .... “Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention...About Viral Vector Elements Learn How to Do Genome-Wide Screens with CRISPR Lentiviral Vectors Additional...

...restriction sites into which you can clone your 20 nucleotide guide sequence of choice (for generation of a gRNA... to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables selection...plasmid post transfection. The 2A "self cleaving" peptide causes the ribosome to translate Cas9-2A and the...the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone. This particular...cleavage site). Finally, in addition to the 2A peptide on its C-terminus, SpCas9 is also fused to a 3X...Resources on the Addgene Website Visit our CRISPR Guide Pages Browse Our CRISPR Collection Find gRNA Design...

... we set out to streamline this process. We take pride in facilitating the exchange of not just the physical...that allows you to conduct your experiments with confidence. Benefits of the UBMTA: Peace of mind Addgene...University Technology Managers (AUTM). This MTA provides many benefits for both the providing and receiving... lists below for more details. Benefits to the provider (organization / lab that owns the material) ...publish without editorial comment or review by the Provider Limited liability Addgene makes it easy Even...can get tricky. Here’s how Addgene helps: For Providers: One-time Deposit: Simply deposit with Addgene...the UBMTA and are happy to agree to it. Their confidence and familiarity with the UBMTA result in quick...

...and stability. TRX and signal peptide tags help proteins with disulfide bonds fold properly. If a TEV ...the subcellular localization and the number of disulfide bonds in your protein - two parameters that affect... and efficient multiplexing. pCri plasmids are divided into two sets, each of which includes plasmids ...designed for these systems may include a signal peptide (SP) that routes the protein through the secretory...secretory pathway to improve folding and disulfide bond formation. If one prefers expression in E. coli, the...thioredoxin A (TRX)-containing vector can improve disulfide bond formation. pCri also includes a special feature...

... Genomics Institute partners with companies to provide technological resources for those studying rare...be used to accelerate rare disease research and provide hope to patients suffering from these devastating...Schiavi plans on using C. elegans as a model to identify SMN1 interacting partners and perform whole animal...of mesothelioma sufferers. This disease is less widely understood and often misdiagnosed. Like other forms...mesothelioma tumor samples that they have used to identify candidate genes as drivers of peritoneal mesothelioma...Meothelioma Cancer Alliance Website.  We are proud to provide support to Drs Di Schiavi and Nabavi and will continue...., et al. "PP09. 1–2352: Systematic review of incidence and prevalence of spinal muscular atrophy (SMA...

...ultimately flattening the curve, it has become evident that widespread testing is crucial in controlling this ...PCR using Taq Polymerase and then transcribed to provide RNA substrates for Cas13. Cas13, upon finding complementarity...RPA, this technique uses Taq polymerase which is widely available and less expensive. Instead of using ..., field-deployable nucleobase detection and identification using FnCas9 (FELUDA) Sensitivity: – 110 femtomolar...FnCas9, this is also a good way to diagnose single nucleotide variations. Also, FnCas9 cleaves in a range of..., field-deployable nucleobase detection and identification using FnCas9. https://doi.org/10.1101/2020.04.07.028167....org Get the basics about CRISPR in our CRISPR guide Browse our entire COVID-19 plasmid collection ...

...Over 25 million people worldwide are currently infected with the lentivirus HIV-1. Today, HIV-1 can be...the virus doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1,...removal of the HIV-1 genome from infected cells, an idea that became much more realistic with the advent ...Cas9/gRNA expression. A pooled analysis found no evidence of Cas9 cleavage at predicted off-target sites...into a therapy, these papers present tantalizing evidence that an HIV-1 cure may be within our reach. Similar... disease that infects over 250 million people worldwide. As we’ve previously seen in the CRISPR field,... Learn to Use CRISPR Pooled Libraries for Genome-Wide Screens Additional Resources onf Addgene.org ...

...different ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning..., I highly recommend watching this entertaining video created by our friends on the Cambridge 2010 iGEM...fragments to incorporate such regions of homology. This video gives a helpful demonstration of how to use Snapgene...the process works best with fragments over 200 nucleotides. This may be because the exonuclease could chew... through an entire fragment shorter than 200 nucleotides before the annealing and polymerization steps...methods (Kahl 2013). However, Gibson assembly isn’t ideal for synthetic biology standards, which rely up on...Type IIS restriction endonucleases) to cleave DNA outside of the recognition sequence. This requires that...

...deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination.) The deletions...Optimizing the system: Brainbow-3 Although Brainbow provided neuroscientists with the vast array of colors ...variety of fluorescent proteins to find those with ideal characteristics (low aggregation, high photostability...produces a minimum of 8 colors. The use of AAV provides spatial and temporal control without the need ...Drosophila and zebrafish. Brainbow in Drosophila has aided in the mapping of neural circuits, such as connections... tool to mark and follow single cells due to the wide array and high stability of colors. Sanes and Lichtman...further refinements of the Brainbow technique will provide important insights into the complicated physical...

...open a heart to look at the inside. We do a lot of interactives and videos, as well, to supplement the...addition to testing those experiments. “We also do videos demonstrating an experiment or a technique to students...gel [for] electrophoresis or doing a dissection video. I did that in January. This involves going in front...the courses encompass what they want to teach.” Besides working with students and faculty, Caitlin also...would be to take any opportunity in the lab or outside the lab: write!” Caitlin did this by editing 15...on Facebook. “I think this helped me say with confidence when I interviewed, ‘I write science. I edit ...

...various conditions. The first thing you'll need to decide when running your experiment, is which vector will...will discuss some of the features you may want to consider as you make your choice. The first and most important...selection marker or antibiotic resistance. Next, consider the expression level of your construct: would ... lentiviral or a retroviral backbone. This is considered to be a more robust expression system and has...are several types of yeast expression vectors. Consider your applications and experimental design. You....1 - TRC cloning vector). Check out our complete guide to empty backbones for additional details and guidance...Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other Plasmid Protocols List of common epitope...

...their interest in science more generally to people outside of academia. Here are my recommendations on some...magazines or newspapers or other places online, and consider how those stories are being told. Twitter can ...science blogs written by practicing scientists. 3)   Consider starting up your own twitter feed or talking about...Close the gap between science and society 6)   Consider participating in a future SciFund Challenge. While...motivated more by the desire to help close the widening gap between science and society by connecting ... Santa Cruz, has been turning scientists into confident and connected science writers since 1981. That... both your plasmids and your research stories. Consider practicing communicating your research by guest...

...interests. We've outlined some important factors to consider and summarize helpful advice from the successful...Achieve Success in Academia". Important factors to consider when choosing a research project After joining...and goals. The following questions can serve as a guide when selecting a research project. What will the... are available in order to realize your project ideas. What mentors are available for guidance, intelligent...fungal biology.” Connie Cepko advises students to consider their goal of PhD training when selecting a research... good judgement with the current technology to identify a problem, asking what approach will I take and...touching a pipetteman. Take the time to generate new ideas and think about them critically before diving in...

... nucleases from re-identifying it (3). Surprisingly, few genes have been identified that neutralize CRISPR...Addgene’s CRISPR Guide or find CRISPR/Cas plasmids for your research. Do you have a cool idea or application... process the CRISPR loci to generate RNA, which guide a Cas nuclease to the specified exogenous genetic...engineering. The diversity of the CRISPR/Cas systems provides powerful lines of defense against invading phages...technology? Let us know.  References Ishino Y, Hideo, S, Makino K, Mitsuko A, Nakata A. J Bacteriol. ...

...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 ...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...structural understanding thereof have the potential to provide improvements to existing technologies based on ...PubMed PMID: 29915061.        A new method to identify proteins occupying a specific genomic locus: Caspex... as an affinity tag. This ‘Caspex’ construct is guided by a pre-defined sgRNA sequence to a specific genomic...preprint posted July 4, 2017 A new method to identify proteins occupying a specific genomic locus: C-BERST... and H2O2, APEX2 labels electron-rich amino acid side chains (e.g. Tyrosine) with biotin. These biotin-labeled...spectrometry allowing a researcher to specifically identify proteins that are enriched in the 20 nm radius...

...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...

...’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...

... 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...

...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...

...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...

...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...

...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...

...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  ...

... 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...

...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...

...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...

...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...

...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...

...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 ...

...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...