...pBR322, pACYC, and pUC were developed to provide higher copy number vectors that could be used in these...updated and added new content to our eBook. Some highlights of the 2nd edition eBook include: new chapters...

...determine genome-wide translational frequency. Although highly useful, this technique requires extensive sample... nucleus to bind cognate untranslated mRNAs. The high specificity and resolution of this system are achieved...SunTag Catch up on Your GFP Background Choose the Brightest Fluorescent Protein Resources on Addgene.org ...

...ask is what to do with them. In some cases, there might be a mutation that disrupts the function of YFG....drivers. It is much easier to mate flies to get the right cell type expression as opposed to making a new ... my next post, I’ll explain the relatively straightforward process of generating your own fly lines. In...

...viral samples. Read on to learn how aqueous samples might be affected by dry ice, and specifically how dry... removing the caps, incubating the tubes in an upright position for 30 seconds, and then recapping the...to prevent any pH change, as we still observed a slight change in pH (from 7.4 to 6.8). It’s possible that...

...as virus? “Like all things in science, getting a high titer Cas9 lentivirus prep takes a bit of time and...lentivirus, we are providing lentiviral preps containing highly requested gRNAs including those targeting EGFP,...library has retained gRNA representation, produce high titer lentivirus from the library, ensure gRNA representation...

...Director of Biology, Lianna Swanson, suggested that I might be able to attend the 10 Years of iPSCs Symposium...or tertiary, or non-existant). On the very first night of the iPSC symposium, a panel of thought leaders...Resources on the Addgene Blog Learn about Efforts to Fight Rare disease with iPSCs Read About Addgene's Outreach...

...but thought we could learn a little bit more about high throughput DNA prepping by trying out NEB’s new ...generating a wide range of insert sizes and also produced high yields of quality libraries from small amounts of...summarize my experience in my first blog post! I highly recommend doing the same if you ever go to a workshop...

...of mutant target (as low as 20 pg or 0.02%) in a high background of wild type targets. Comparatively, ...M. A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells. PLoS...PubMed Central PMCID: PMC4735761. 6. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas...

... Directors brings complementary experience and insight to the now 10 member board at Addgene. Let’s meet...Suffolk University Law School. Karen graduated with highest honors from the University of Michigan, Ann Arbor...Ernst & Young, LLP, auditing insurance, finance and high-tech industries. He has earned both an MBA in finance...

...this context, but also specifically study it. They might be researching enhancer elements that control the...research describe the production of plasmids that might be useful for your work. The links below direct ...techniques, the posts below may steer you in the right direction! CAPTURE-ing Chromatin Interactions: Using...

... synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a T7 promoter driving multiple...GAL1 promoters. These vectors are intended for the high level expression and purification of SARS-CoV-2 ... strengths of intensive screening and the use of high throughput platforms, Addgene and Ginkgo are striking...

...followed by higher stringency PACE, resulted in the evolution of TadA variants with much higher base editing...simultaneously with 54% efficiency, about 7 times higher than ABE7.10. However, the increased editing speed...

...narrow down search results by adding more terms.  Weighting We determined the relative importance of different...given resource, and designed the search engine to weight search results differently according to where a...matches of a name in the depositor field will rank higher than matches of the same name in the publication...

...complicated? Well, while the techniques are straightforward, the systems – the cells growing and producing...And on top of that you need to worry about getting high enough expression of your protein of interest. If...by using Addgene’s Blue Flame award to identify highly requested GCE plasmids. But they’d also love scientist...

...variant that efficiently infects neurons, directs high-level transgene expression, and has retrograde functionality...genetic tool delivered by the AAV.   AAVrg requires high titers for efficient retrograde transport. For recombinase-dependent... at the injection site (Fischer, KB. 2019). The higher the titer, the more likely you are to see spurious...

...their former interns to fill entry-level positions right out of college. Prior to March 2020, internships... a nationwide database of  STEAM internships for high school, college, and graduate students. In order...interview, inspect your technology, background, and lighting for a smooth performance. Most importantly, make...

...). As shown on the representative plate to the right, colonies with an insert-containing plasmid have... plates at 4C for few hours after the initial overnight incubation increases pigment precipitation, enhances...colonies. Take care in making your plates: X-gal is light and temperature sensitive and needs to be added ...

...interested in the same things you are on a deep level. Right away you have something in common. Namely, the scientific... field will know about grants and projects that might help your work Tenure can depend on your national...some of my best studies were designed over late night poker games at Gordon Conferences). You don’t want...

...zygotes at 18 hr post fertilization. They observed higher targeting efficiency than in iPSCs, but with the...scientists proceed with caution and with proper oversight from regulatory and ethics committees comprised... Check out Our New CRISPR Topic Page Read about High Fidelity CRISPR Variants Target Multiple Genes at...

... read an 11-page Cell paper - and sometimes, it might actually take me less time to read the book.  Intuitively...science communication, but the more you use, the higher cognitive load you are asking your audience to ...like the professor who once decided that to teach high school students PCR, they first had to understand...

...antibodies that produce detectable signals like light or color. In this post, we’ll go over some of the...chemiluminescent reaction. Chemiluminescence, which is light produced by a chemical reaction, can be detected...Whatever the conjugate, the molecules must somehow be tightly linked to your antibody. This chemical coupling...

...working with an endogenous locus. The most straightforward way to turn off expression permanently is to...- this section is for you! Many of the tools highlighted in this section can be used to turn expression...

...have less complementarity to the gRNA, they have a higher propensity to reform double helices and decrease...measurements to determine which portions of Cas9 might be important for nuclease specificity. They found...PMC4176945. 4. Kleinstiver, Benjamin P., et al. "High-fidelity CRISPR–Cas9 nucleases with no detectable...

..., TdTomato was seen only in cells with GFP, at a high activation frequency of 56-93%. In the converse ...Cre-DOG lacks the toxicity that can be seen with high levels of transcription activation domains. Both...constructing split variants should be relatively straightforward, but it is also possible to create split versions...

...chronic myeloid leukemia. Bhang says the work highlighted the importance of developing more potent ABL1...clonal dynamics in response to cancer therapy using high-complexity barcoding." Nature medicine 21.5 (2015...

...launched an online career coaching program that might make her coaching more affordable for you. There...transitioning a scientist from academia to industry, you might want to consult the Cheeky Scientist. Their motto...Successful Industry Professionals”. This larger shop might appeal to some although most scientist coaches can...

...understanding of cell biology. We hope that these high-quality, highly specific plasmids, as well as the instructions...flooding the cell with a plasmid containing regions of high homology to the gene with the cut, we trick the ...

...company. This might seem like a no-brainer, but it happens more frequently than you might think. The Addgene... crucial information about a product is printed right on the tube itself! Is the answer to your question...

...endogenous retrovirus Rless glycoprotein (BaEVRless) had high rates of transduction for most cell types they tested...existing CRISPR delivery technologies, Nanoblades had higher rates for editing efficiency and lower rates of...zygotes, which results in albinism in mice. Of the eight mice born from Tyr Nanoblade treated zygotes, five...

.... In the first blog post in this series, I’ll highlight terminology you should be familiar with before...the use of long single-stranded DNA have led to a higher efficiency of homologous recombination for precise...

...undergraduate research questions. The idea is straightforward. Instructors first select a multi-gene pathway...O'Connor (left with Lab Director Doug Warner on the right) has taught courses in biochemistry, molecular cell...Addgene Videos to Learn about Basic Lab Techniques High School Students Explore Careers at Addgene Resources...

...host) as well as the introduction of the cancer-fighting component, CAR. Clinical trials are underway to...allele specifically. Early-stage trials showed a high success rate in clearing misfolded protein levels...Regarding effectiveness, if the editing frequency isn’t high enough to make a physiological difference, then ...

...ER lumen reaches the sub-millimolar range, much higher than in the cytosol. Thus, for the ER you would...subsequent change in fluorescence only occurs at high calcium concentrations. This will reduce noise and...simultaneous (1) user-controlled illumination with violet light and (2) exposure to increased intracellular calcium...

...recombinase systems. Picking Ligands Picking the right ligand is also important for building a chemogenetic...pharmacologically selective effector molecules (PSEMs). Higher potency means lower drug doses, which decreases... CH, Coward P, Degtyarev MY, Lee EK, Kwa AT, Hennighausen L, Bujard H, Fishman GI, Conklin BR (1999) Conditional...

...lucky" and find a great gRNA with a proximal PAM right where you need it, the odds of this occurring are...reliability. Notably, the SpRY variant didn’t display higher off-target effects than SpCas9, a pleasant surprise...stranded cleavage and base editing applications, with high editing capacity in both contexts for NRN PAMs and...

... of interest thus represents a straightforward means of exerting tight control over Cas9 nuclease activity...different genetic loci within a single cell. One might imagine transfecting cells with both the nuclease-positive...

...something silly like “See there, arginine 53 really is right there, at the end of helix 3…just like we said it...are useful. While a physical model of a protein might represent one aspect of the protein very well, it...while and you will wake up in the middle of the night dreaming about the conformational gymnastics that...

...primers.  Extension- The temperature is then raised slightly to Taq polymerase’s ideal temperature (70-75oC...which can be time consuming and challenging to get right. ddPCR uses a water-oil emulsion droplet technology...those stated above. Multiplex PCR is also used in high throughput SNP genotyping, gene deletion analysis...

...colonies by (carefully) holding plates up to the light, where they appear as opaque dots (colonies should...the agarose onto the recipient plate at just the right temperature. If the agarose solution is too hot,...depending on the level of endogenous expression. We highly recommend the use of an electronic pipettor with...

... Sometimes people don’t know how to start. They might want to use our promoters and terminators, but then...coding sequence to the MoClo system. It’s quite straightforward really and we’ll be producing an instructional...chances of doing something amazing will be equally high as for those who go to work in cancer biology. ...

...to make the most of your time and sequence! You might also enjoy seeing how our quality control process...alignment results. The default setting is “Optimize for Highly similar sequences (megablast)”, which is very fast...nucleotide sequences, proteins often have repeated or highly homologous regions, which by default are ignored...

...a collection of promises to not assert property rights for particular engineered genetic sequences; but...metabolite in a cell. The only way to do that at scale right now is to grow up a bunch of cells, sacrifice them...chemicals, to make more medicine. The parts we have right now are well adapted to work in prokaryotic bacteria...

...available Not commercially available Small but Mighty: Advantages of Nanobodies Although antibodies have...target antigen to about 2 nm, which can lead to higher resolution for super-resolution microscopy. Nanobodies... two questions, refer to this table to find the right nanobody for your work. Species Antibody Subclass...

...standard curve for qPCR can be challenging to get right. Here at Addgene, we use a linearized plasmid standard...very few positive droplets. In the image to the right, there is approximately 1 positive copy per microliter...droplet generator to the PCR plate. Maintaining a high droplet count is important for calculating the concentration...

...trainees’ very way of thinking too. Albert Einstein might have put it best when he stated, “the value of an...pathways (precisely the type of exercise Einstein might have resented!). In 1997, Joseph Provost, then a...like an exciting opportunity for your classroom, I highly recommend you try implementing one. It's likely...

... know the basic principles of IF, we’ll take a slightly deeper dive into those steps and provide you with...commonly used cross-linker for IF. However, you might come across a couple other terms that can cause ...especially if left on for too long or used at too high a concentration.      Figure 2: Examples...

...A) Streamlined VchINT expression constructs. (B) High integration efficiencies achieved with VchINT, at...mini-transposon Transform plasmid(s) into cells and plate overnight Screen colonies phenotypically or by PCR/sequencing...Sternberg SH (2020) CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering...

... off-target effects. Cas9 RNPs are detectable at high levels shortly after transfection, and are quickly...efficiency, while biolistic bombardment, which can have high efficiency, requires specialized equipment, such...microbiology.     References 1. Chen, Sean, et al. "Highly efficient mouse genome editing by CRISPR ribonucleoprotein...

...interested audiences quickly and concisely – video might be part of the answer. Problems with written publications...TedTalks are very popular, though they focus mostly on highly innovative ideas rather than on the day-to-day ...that provides links to the videos, keeping the copyright and control over the videos with the researcher...

... #MolluscMonday: #Crepidula atrasolea juvenile, light microscope with polarizing filter. Development is...an extreme number of nerve cells and an unusually high level of RNA recoding in the brain. RNA editing ...DNA in the eggs to get transgenic animals. They might be immortal. Manihot esculenta (Blake Meyers) Cassava...