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...protocols for plasmid cloning Find our molecular biology reference here Find molecular biology protocols...bacterial recombination pathway, referred to as recA-independent recombination, joins together pieces of linear...sequences! Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while modifications...encoded at the 5’ end. First design the template binding region, with a melting temperature (Tm) of around... as epitope tags, design primers with template binding regions to amplify away from the insertion site... To remove part of a plasmid, order primers that bind at either side of the undesired portion, designed...

...expression in mammals Resources at Addgene.org Find AAV plasmids Find ready-to-use AAV viral preps Browse all...horribly wrong. It brought the whole field to a grinding stop and only now, 2 decades later, is it finally...autosomal recessive, severe, childhood-onset form of blindness name Leber’s Congenital Amaurosis (LCA). Shortly...disease that results in combined deafness and blindness) is caused by defects in the gene MYO7a, at ~7...further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the...restores vision in a canine model of childhood blindness. Nature Genetics 28, 92-95. PubMed PMID: 11326284...

...our Fluorescent Protein Collection Find Plasmid Kits at Addgene Find Plasmid Cloning Protocols References...promoters, enhancers, polyadenylation signals, inducible gene-expression sequences, and more. By combining...constructs to visualize cell cycle dynamics, titrate inducible transgene expression, or a variety of other clever...label subcellular structures in live cells. Four individual expression constructs were created, each containing...Repeatable, directional cloning   The principle behind MXS-chaining is repeatable, restriction-enzyme ...cloning. The modules (Figure 2) are contained in individual plasmids and are each flanked by the same multiple...

...the learning of many facts but the training of the mind to think something that cannot be learned from textbooks... (Brian, 1996). But is it possible to bring the kinds of transformative educational experiences afforded...happened in the lab, and he wanted to provide the same kinds of opportunities for his students. But he also found...genuine interest to other researchers in their field. Indeed, sharing results with the broader scientific community...interact with citrate synthase and presented their findings at the American Society for Biochemistry and Molecular...research, they gain confidence and learn to work independently, which can lead to greater success in meeting...working, hypotheses being rejected, etc.), while reminding participants of the value of this approach and...

...on Addgene.org Find CRISPR Plasmids for Your Research Read Our CRISPR Guide Pages Find CRISPR gRNA Design...bacteria causing an infection, but also alter an individual’s native, beneficial flora. Currently, there is...-homologous end joining machinery, so a CRISPR-induced double-stranded break (DSB) in the genome is lethal...be eliminated from the bacterium, which may also induce cell death. CRISPR also enables increased specificity...sequence-specific, antimicrobial, plasmid-based, CRISPR systems (Find the Citorik Plasmids here). In both cases, they ...PMID 252409288. PubMed Central: PMCID PMC4˚237163. Find plasmids from this paper at Addgene. 2. Bikard,...

...Learn about Addgene's viral service Find viral protocols and videos Find AAVs for your research ...Locomotion is a highly complex process on the level of individual neurons and in terms of their interactions with...like stress levels, but researchers have to be mindful of motivation overlap. The locus coeruleus (LC)...2015) used optogenetics to support these existing findings by controlling neurons in the mPFC with light....depression, yet there have also been contradictory findings (Groves, 2007). A study from 2014 (Bahi et al....Addgene blog Read all of our viral vectors blog posts Find tips for getting started with viral vectors for ...

...organisms - organisms have codon usage bias. You can find many codon tables showing which codons encode which...preferences and what can we do about them? Read on to find out!   Why do organisms have different codon usage...instance, it may not express at all in E. coli (you can find a variety of databases showing various organisms...codon choice may interrupt transcription factor binding sites) As you might imagine, it’s not easy for...GenScript host online codon optimization tools. Keep in mind that, just because you optimize a gene with one ...et al. "Selective charging of tRNA isoacceptors induced by amino‐acid starvation." EMBO reports 6.2 (2005...PMCID: PMC552224. 4. Gustafsson, Claes, Sridhar Govindarajan, and Jeremy Minshull. "Codon bias and heterologous...

...priorities to attend to. Being mindful looks like: “When you have time, would you mind helping me with….” vs. ...become proficient in lab techniques, which will hinder your progress. You will also probably have a lot...there enough information there for you to be independent performing the technique you trained to do? Walk...clicking’ for them. It’s often easiest to train individuals who are polite, appreciative, and clear about... be done by the researchers themselves.  Be mindful that your mentor will still have their own deadlines...Have a Mentor, What Now? Will You Be My Mentor? Finding and Asking for Mentoring Support   ...

...clients. In this realm, there are a few different kinds of challenges that consultants get called upon to...healthcare consultant, I have worked on most of these kinds of projects. Almost all of my projects, have involved...commercial future, and yet are incredibly difficult to find solutions to. There are many reasons why client ...particular therapy. With your scientific knowledge behind you, you’ll have the opportunity to learn the business...those conversations, reading online healthcare industry publications like FiercePharma, FierceBiotech,...about the trends and workings of the healthcare industry. Finally, on-campus resources like career offices... both provide up-to-date information about the industry, recruiting processes, and can connect you to ...

...your own, but if you ever find yourself in need of a few ideas, I hope you’ll find this post useful. After...this, I always make sure I have explicit goals in mind for the endpoints of a task or project (with metrics...hand write to-do lists at the start of each day to remind myself of the specific activities that I need to... better when I write them down. Some people also find it useful to write out their to-do lists at the ...handwriting, is copied below): Read new Zhang Lab Paper Find plasmids similar to those in the paper Ask Mary ...everything you need to do by 6 pm. Hopefully you’ll find this post useful for making your days, experiments...

...some creative approaches to lab sustainability, and find ways to further reduce our consumption beyond recycling...energy, and water use. ACT labels are obtained by independent audit, and companies need to pay to obtain them...grades of plastic lumber and distribute it to industrial and residential markets. Rethinking our approach...practices to meet higher environmental standards. Find some easy action items on our other article in this...initiatives for the lab Subscribe to the Addgene blog Find lab tips for your research Resources on Addgene.org...other scientists save resources by sharing plasmids Find plasmids at Addgene ...

...to Addgene to find the plasmid or viral vector for your next experiment, that you can find what you need...finally share with you what we’ve been working on behind the scenes here at Addgene.  Addgene's new homepage... content was either small, cluttered, or hard to find. This made the page look not only confusing, but...browse functionality. We wanted users to be able to find plasmids and viral vector tools more easily. In ...what Addgene has to offer, and to help scientists find what they need - whether it's their first or 500th...

...Addgene.org Find CRISPR Plasmids for use in Mammals Check out Our CRISPR Guide Pages Find Plasmids for...have been accumulated over the years using them. Indeed, the future of mouse work is bright as it is now...if we could just go in and quickly manipulate individual mouse genes and study their function in health...this experiment showed a mutation! I looked out my window and I saw the Dorr Mountain with the rest of the...-homologous end joining (NHEJ), this will leave behind a “scar”, with nucleotides missing or added at ... when generating knockout models with CRISPR and find that many, if not all, the mice carry a frameshift...

...activated with light.  Find plasmids from the Isacoff lab! They then tested what kind of swimming behavior... ready-to-use viral service Find optogenetics AAVs for your research Find all AAVs from the viral service...even be expressed simultaneously and still be individually activated with different lights (Baier et al...PMC2770190.  Additional resources on the Addgene blog Find all our optogenetics blog posts Read blog posts ...

...Both D10A and H840A are potent editors, but their indel profiles vary. In the PAM-out configuration, D10A...nickases for HDR is targeting range: using an individual gRNA with WT Cas9, repair levels decrease rapidly...rapidly 10 bp from the cut site. So if you can’t find a good gRNA that cuts close to your insertion site...throughout the entire region between the nicks? To find out, Schubert and Yan designed a PAM-out nickase... to using WT Cas9 with the left gRNA across the window. In a subsequent experiment, they were also able...Resources on Addgene.org Check out CRISPR Topic Page Find CRISPR Nickase Plasmids Browse the CRISPR Guide ...

...initiation of a vast number of signaling cascades, inducing different cellular responses. One of the biggest...complexes that are activated in response to a ligand binding a GPCR of interest. How does TRUPATH work? The ...bound to the intracellular surface of the GPCR. Binding of a ligand to the extracellular surface of the...bioluminescence resonance energy transfer 2, or BRET2. With binding of the drug, the alpha and beta subunits dissociate...Browse all of our fluorescent protein blog posts Find other fluorescent biosensors Resources on Addgene.org...Browse the catalog of fluorescent protein plasmids Find biosensors for your experiments ...

...it easier for you to find and use the plasmids you need. Here’s what you’ll find in this article: CRISPR-based... generation, controllable gene knockouts, and inducible gene inactivation or activation in an organoid...Organoid EasyTag system, showing the workflow for inducible CRISPR inhibition (CRISPRi). A) Lentiviral vector... to the petunia SL enzymatic receptor DAD2. SLs induce a conformational change of the receptor which modulates...plasmids are always being added to the repository. To find all of the CRISPR plasmids available from Addgene...

...   NEBCutter2: For finding restriction enzyme cut sites Webcutter 2.0: For finding restriction enzyme ...enzyme cut sites Open reading frame finder  Addgene vector database: Find sequences for plasmids assembled ...Reagent and Data Repositories These links can help you find experimental and/or validation data on molecular...Addgene.org: Read our Molecular Biology Reference Find molecular biology protocols Browse our protocol ...

...of vectors for inducible expression (pSBtet derivatives). The constitutive and inducible expression vectors...transposase enzyme is expressed (red stars) and binds the inverted repeats (green); and an endonuclease...cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there are...Jan;257(1):181-90. doi: 10.1111/imr.12137. PubMed. Find the Plasmids at Addgene Kowarz Lab plasmids Izsvak... inducible vectors...

...and published by academic scientists. To help you find the vector that fits your experiments, I've described...close as possible to the basal expression level, induce expression at an opportune time or go all out with...In most cases bacterial expression vectors are inducible (e.g. pBAD LIC cloning vector (8A)) and have an...be released from the resin and used for various binding or folding assays. Yeast vectors There are several...integration into the fly genome and are under the Gal4 inducible UAS promoter. Other vectors can be expressed in...the comments below and an Addgenie will help you find what you need.   More Plasmid Resources from Addgene...

...Allele Biotechnology, Florida State University, Indiana University, and the Karolinska Institute developed...immunoprecipitation experiments with other fluorescent proteins. Find mNeonGreen plasmids here! mTFP1: a monomeric teal...insensitive to physiologically relevant changes in pH. Find mTFP1 plasmids here! mWasabi: a bright green fluorescent... significantly excite at 400 nm while EGFP does. Find mWasabi plasmids here! One of the most notable things...Addgene blog Read our fluorescent protein blog posts Find blog posts about choosing fluorescent proteins for...Addgene’s fluorescent protein plasmids and resources Find fluorescent protein kits ...

...training progresses.  In From Academic Solos to Industrial Symphonies, an article well worth reading on ...can directly interfere with a seamless jump into industry.” In his article On Leaving Academe, Terran Lane...excellent—some say “cushy”—tenured faculty position for the grind of corporate life”.   In fact, there are a lot of...budget constraints Academic science is much like industry but the “product” is publications—the pressure...—science can be surprisingly more rigorous in industrial settings Tight (sometimes impossible) timelines—others...training and resources within the company Must find own sources of knowledge often externally A bit...

...not overgrow the plasmid at 37°C. Some researchers find that using a 30°C incubation temperature to grow...vector backbone vs. the full length plasmid. If you find that your bacterial cultures contain a mix of full... bands: nicked, linear, and supercoiled. Here we find a smaller, recombined plasmid around 1.5 kb. When...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full....069286.118 Additional resources on the Addgene blog Find more molecular biology and viral vector protocols...protocols Learn about the common lab E. coli strains Find tips for viral transduction  ...

... previously. While this claim has yet to be independently verified, the watching world erupted in controversy...private funder, government regulators, and an independent ethics council – all worked in tandem, within...experiment on humans, and they are not remembered kindly. However, despite many headlines questioning if...utilizes her firsthand experience in the software industry to inform her work on emerging issues in novel...theirpenmanship. Additional resources on the Addgene blog Find all of our CRISPR blog posts Download the CRISPR... Resources on Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Check out our genome...

...Resources on Addgene.org Find molecular biology educational resources Find protocol videos to teach basic... and understand how therapeutic strategies work. Find Addgene Blog posts about other educational tools...Accessing and modifying BUILD YOUR PLASMID You can find rules, suggestions, and more details on how to play...FRE_SCI.   Additional Resources on the Addgene Blog Find planarian tools for teaching developmental biology... can help teach students about Synthetic Biology Find Arabidopsis educational resources at the Arabidopsis...

...Additional Resources on Addgene.org Find Ready-to-Use AAV Preps Find Lab Protocols for Work with Virus ...titers Making contact with a cell Although an individual AAV particle’s ability to infect a cell is dependent...concentration. In this flowchart, you can see the dwindling proportion of AAV particles that make it through...high titers, as contact between the cell and any individual AAV particle is rare. But simply getting into...higher titer you will have. When possible, use low-bind or siliconized tubes to store samples as it is more...

... at 230 nm. The A260/A280 ratio is used as an indicator of DNA purity. Ideally, this number should be ..., you can use the agarose gel method to not only find out how much DNA you have, but also to see whether...like a PCR product). This method also gives an indicator of DNA or RNA contamination based on the presence...no other instruments are available. Some last reminders about quantifying DNA In choosing your DNA quantification... more about molecular biology protocols and tips Find the best way to elute and store your plasmid DNA...Addgene.org Visit the Molecular Biology Reference Find plasmids for your research Browse protocols for ...

... phosphorylates a response regulator (RR). TCS-induced responses are varied; one outcome is directed transcription...promoter strength of various components, removing inducible promoters that could crosstalk with other pathways...doi: 10.1021/sb500273n. Epub 2014 Oct 11. PubMed. Find the plasmids from this publication at Addgene. ...CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find the plasmids from this publication at Addgene. ...1272-81. doi: 10.1016/j.cell.2009.04.048. PubMed. Find the plasmids from this publication at Addgene. ...10.1016/j.jmb.2010.10.038. Epub 2010 Oct 28. PubMed. Find the plasmids from this publication at Addgene. ...

... passed down through cell division. You can find all kinds of different lentiviral and AAV plasmids as...protocols for plasmid cloning Find our molecular biology reference here Find molecular biology protocols... of a fine needle to deliver nucleic acids to individual cells. Electroporation on the other hand uses...edition (2000). Meibom, Karin L., et al. "Chitin induces natural competence in Vibrio cholerae." Science...

... and Immunity and learn more about “... recent findings in basic immunology, cancer-immune cell interactions...discussed the effects of starvation on cancer therapy. Findings in previous animal studies suggested that fasting...in trying to resolve the inflammatory state by inducing immunosuppressive measures, for example the upregulation...impressive videos of these interactions (Ref 3). You can find useful tips on in vivo imaging in our Fluorescent...scientific community, contact [email protected] to find out more about our free deposit and distribution...Browse Cancer Plasmids Browse Immunology Plasmids Find Kits for Cancer Research  ...

... an individual to another individual perspective? A: CAP-B10 and CAP-B22 were tested individually in 4...question that didn’t get answered live - but he kindly took some time to address all your unanswered questions... 4 marmosets each. Brain potency for each individual is reported in Figure 4B (see left for image and ...a first time AAV user Resources on Addgene.org Find Adenovirus Plasmids Adenovirus Guide Page Subscribe...

...able to find strategies and systems that set you up for success in the detail-driven daily grind of science...tedious, but it also works, so do it anyway.  You may find that after running a protocol a few times, you no... blot protocol, from a full page to a few key reminders on a sticky note   Visual Planning  Plate ... use cold block systems or buckets of ice. Try finding ways to intentionally set up your bench to help...forth, continuing all day or even all week. By finding ways to intentionally slow down, you’re giving ...

...synthetic beads that can bind to a conjugated antibody and beads that are unable to bind to them: like cells...real results from background noise or nonspecific binding. Flow specific controls The three types of flow...they are non-specific. The compensation beads can bind to any primary antibody you add. Single color controls...allow you to determine what level of non-specific binding you have in your sample. The isotype antibody will... This enables you to identify any non-specific binding to know that your positive cells are truly positive...

...of interest, as it is prone to generating indel errors. Indel errors generated in the course of repair ...where mutations are introduced. Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated...microhomologies”). NHEJ doesn’t obligatorily introduce indels. Given the end structure of the Cas9 DSB (blunt... of accurate repair are easily re-cleaved while indel products aren’t, so repeated cycles will favor accumulation...constitutively expressing a targeted Cas9 should possess indels in the majority of their chromosomes within a day...

... at multiple levels to generate anything from individual transcription units (TUs) used to express your...metabolic pathways. For many MoClo systems, the individual parts are reusable, saving time and money on ...some common MoClo applications and kits. Keep in mind that this technology is useful for a variety of ...purification tags, connectors, cDNAs, and more to find the perfect combination. The EcoFlex Kit has a wide...array of DNA parts that can be assembled into individual TUs or very large (up to 20!) multi-TU constructs...et al., 2016). The variety of constitutive and inducible promoters, linkers, and tags makes EcoFlex a great...kits are designed with specific model organisms in mind, others are designed for flexibility. The Fragmid...

...qualify what kind of “plate” you mean. Without context, it’s difficult to know what kind of plate they...figure out the sources of miscommunication and b) find practical solutions.  My PhD prepared me remarkably...document, I update the training frequently. In hindsight, getting a list of frequently used words like ...Addgene blog Read about other transferable skills Find all of our science career related blog posts Read...communication blog posts Resources on Addgene.org Find careers at Addgene ...

...established, it’s also a process that’s difficult for individual labs to complete. The nanobody based RANbody ...purification. Antibodies are difficult for an individual lab to produce - Hybridomas offer an alternative...RFP,  the histone protein H2A2B, and the actin-binding protein gelsolin, but any nanobody sequence could...serves as a backup means of detection that’s independent of the reporter. The only caveat is that HA staining.... Staining with multiple HRP-RANbodies is indistinguishable since they produce the same signal, so only...plasmids from Yamagata et al can be found here. Can’t find a RANbody against your favorite protein? No worries...

...system, antibodies are produced by B cells. Each individual B cell produces antibodies that all recognize...purification is one common method. These proteins bind the IgG heavy chain. Running the serum over a column...column of resin crosslinked to protein A /G will bind the IgG antibodies present in the serum, whether... are isolated from the serum by their ability to bind the target antigen. In this case, the serum is run...antibody; the ability of polyclonal antibodies to bind many epitopes of their target protein thereby greatly...polyclonal isotype control, to account for nonspecific binding. Now that you understand the pros and cons of choosing...

...2013). We found that the fluorescent proteins can, indeed, interfere with the correct function of the protein... species (bottom panel in Fig. 1). Our results indicate that the tag protein may have an important influence...experiment. Since the fused protein could represent a hindrance to the successful expression of the protein of...opsin-expressing cells). For other things to keep in mind when making fluorescent protein fusions, check out... posts Read about chemogenetics vs. optogenetics Find more about the many types of protein tags References...Addgene.org Check out Addgene's Chemogenetics Guide Find fluorescent proteins at Addgene ...

...next week!”, and “This is his job!” filled my naive mind. Moving forward from that first experience, I learned...process, she has faith in you! Additionally, keep in mind that universities require application submission...the mission of the agency and benefits the field reminds the reviewer why your project is the project to...travel, etc. As a first time budget writer, you may find this section challenging because it’s hard to determine...students and postdocs will be hired, direct and indirect costs, etc. It may be helpful to ask your advisor...application process? What types of writing feedback do you find most helpful? Have a great grant writing tip? Share...