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...together DNA fragments. As a fun way to start, I highly recommend watching this entertaining video created...assembly work? The required homology between neighboring fragments can be created via PCR amplification...including regions of homology to the vector or neighboring fragment. Then you would amplify the fragments...success rate of this reaction is typically fairly high, so there is usually no need to screen a large amount...structure, such as a hairpin or a stem loop (as might be anticipated to occur within a terminator sequence...required single-stranded annealing and priming of neighboring assembly fragments. Gibson assembly is commonly...vector that you want to use is very large, has a high GC content, contains a lot of repeats--any of which...

...via gene therapy, so researchers have set their sights on another approach. Since shorter forms of dystrophin...to persist over time, stem cell editing would be highly desirable. Should stem cell editing occur, the ...and AAV9, respectively. Although each study used slightly different methods, they each observed the same..., the relative abundance of exon-skipped mRNA is high, likely because this mRNA is not subject to nonsense-mediated... very low to no off-target activity at the ten highest-ranking predicted off-target sites. Each paper ...CRISPR delivery must be optimized to: 1. Reach a high percentage of muscle cells throughout the body, ... rigorously examined over a long period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should...

...Addgene: sharing speeds science. Here, we’d like to highlight the advances in base editing that made this treatment...of KJ’s CPS1 mutation. Every genetic target is slightly different, so more options mean better chances... turned out to be most effective. Targeting the right PAM Expanding PAM preferences Base editors can only... only access a short sequence, between four and eight bases, close to the PAM recognized by Cas9. If no...target many sites in the genome, they also have a higher risk of off-target editing and longer genome search... ABE8e performed the best when paired with the highest-efficiency sgRNA. The PAM sequence near this site...N. A., Small, J. C., McCague, S., Burke, S. M., Wright, C. M., Bick, S., Indurthi, V., Sharma, S., … Ahrens-Nicklas...

...transcribe the extremely long, highly structured HCV genome (~9.6 kb of highly structured template!). MarathonRT...Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...Listen to the Hot Plasmids Podcast!  MarathonRT: A highly processive reverse transcriptase Article contributed...complex heterogeneous RNA samples. Furthermore, such high processivity enzymes and the structural understanding...antibodies: they are small (12-15 kDa), stable, highly soluble, and non-immunogenic yet are still just...

...Syndrome One of those teams is led by Claudio Aguilar (right) at Purdue University and is working to understand...forward. Xeroderma Pigmentosum Hendrik De Buhr (top right) and his colleagues at the Wistar Institute in Philadelphia...condition leaves patients incredibly sensitive to sunlight and prone to developing metastatic melanomas and... and other cancers. Joshua Wang (bottom right), also of the Wistar Institute, says that the lab got interested...Alternating Hemiplegia of Childhood Melodi McSweeney (right), a graduate student at Duke who has recently moved...returning to basal conditions normally after periods of high activity. The new work will help them to understand... Syndrome Last but not least, Meng-Shin Shiao (right) of Mahidol University’s Ramathibodi Hospital in...

...copies can be tightly controlled by titrating the transposase to transposon ratios. High ratios of transposase...efficiency of excision with PBx has been shown to be higher than that of the wildtype piggyBac™ transposase...importantly, unlike Cre recombinase, the PBx is highly specific, and has not been shown to cause genomic...specialized knowledge to help researchers identify the right technologies and strategies for their projects. ... 2016. PubMed PMID: 27075976. 4.  X. Li, E. R. Burnight, A. L. Cooney, N. Malani, T. Brady, J. D. Sander.... Qualls, B. M. Lewis, E. Ke, C. T. Dargitz, R. Wright, A. Khanna, F. H. Gage, and I. M. Verma, “Functional..., M. van Lohuizen, A. Berns, and D. J. Adams, “A high-throughput splinkerette-PCR method for the isolation...

...tricky at best, and downright infuriating at worst. While it is tempting to just dive right in and start injecting...also transduce postsynaptic cells when used at very high titers (Zingg et al., 2017), which can lead to cells... total injection volume. You’ll often hear that “high titer” is required for efficient AAV transduction...the spread of the virus.  A better way to define high titer, then, is this: It’s the optimal number of...inversely related to transduction efficacy: the higher the efficiency, the lower the titer and vice versa...needed volume (de Backer et al., 2010).  Second, high efficiency transduction coupled with a strong promoter...amount that looks effective. Ultimately, the perfect “high titer” is one that optimizes gene expression where...

...promoters, and even modest endogenous pools generate high signal-to-background luminescence (Schwinn et al...tags floating around the scientific world, so you might wonder what sets HiBiT apart. While the classic ...weak signals; photobleaching from the excitation light, causing the signal to gradually diminish over time...vesicles (EVs), edited transcripts could endow neighboring cells with pro-oncogenic traits, facilitating...spread to enhanced malignant potential. This work highlights HiBiT tagging as a sensitive reporter for tracking...HiBiT provides an excellent solution compatible with high throughput processing and enables a wide range of...try and see your favorite proteins in a whole new light!   This post was written by Simon Moe, an ...

...fold increase in brightness after ~100 action potentials. This is about twice as bright as GCaMP7f and ...action potential, GCaMP7s is approximately twice as bright, relative to baseline fluorescence, as the GCaMP7f...GCaMP7f variant, and is approximately 5-fold brighter than GCaMP6s. jGCaMP7f is the fastest responding variant... variant. jGCaMP7b exhibits the brightest resting fluorescence and can be used for imaging small neuronal...processes (dendrites and axons). jGCaMP7c exhibits high contrast between peak fluorescence and resting fluorescence...lowering sensor resting fluorescence, while maintaining high peak fluorescence) and is useful for imaging signaling...

...specific goal, and when you realise that your goal might not lead to a career you actually want, you can ...applicable to a wide range of industries and is a highly desirable quality sought after by employers. These...many employers. Increasingly, computer skills are highly desired, as roles are becoming more reliant on ...Programming skills (Python, R, SAS, HTML, Matlab etc) are highly desirable in a range of roles, including: Science...give you an idea of what additional training you might need to obtain. You'll also need to think of other...you have the relevant skills and experience.You might not normally think about it, but hobbies and volunteer... job involves traveling, showing that you are a highly organised and experienced traveler by citing specific...

... in Paris and New York. We want YOU to join the fight against coronavirus We are just at the beginning...be the next scientist joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in...that we have to stick together (6 feet apart), to fight the new Coronavirus. To help get you started, here... sites - so cloning projects should be fun and straight-forward. All constructs are tagged with a 2xStrep-tag... gave a lot of background in our study - so you might want to be careful with these clones (and help us... research. For all these reasons, the QCRG is delighted to be partnering with Addgene, who will be taking...bloggers Manon Eckhardt (left) and Melanie Brewer (right) from the University of California, San Francisco...

...convince your potential employer that you are the right candidate for the job. How can you incorporate the...you need to have a good idea of what should be highlighted on your resume, and tailor the resume for the...programs to predict protein structures, then you might know a thing or two about computer modeling. Make...transferable skills Some of the skills you developed might not have come from the research itself, but from...universities actually have career services offices. You might think only undergraduates use these services, but... work well with your life and career plan? This might be a tough question to ask now, but the earlier ...

...standard) and reflects a desire for very high MOI on top of very high coverage. Please see this paper for ...that this will require co-delivery of two AAVs at a high MOI, often resulting in a drop in transduction coverage...particles into the same cells in vivo? A: This will be highly dependent on vector dose. The color diversity in...experiments using PHP.eB in the brain give some insight into codelivery efficiency at different doses. ...liver transduction. While we have not tried it, it might be worth investigating for both Q6 & Q7.For epithelial...that we have in house!), which seems to me a very high dosage. Any thoughts?  A: The question refers to...

...and 31 times higher, respectively. In rice protoplasts, Xie et. al observed slightly higher mutation rates...this system is clearly also robust enough to ensure high levels of gRNA cleavage.   Xie et. al constructed...nucleotides to the 5’ spacers. The 3’ ends were modified slightly with either a 1-7 base poly(U)tail in tRNA-gRNA... had biallelic mutations at all five loci. This highly efficient multiplexing further illustrates the ... Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Tsai SQ, Wyvekens N, Khayter...

...plasmid may be expressed at higher than normal physiological levels due to high copy numbers. This creates...isolation, a well-defined genetic system, and a highly versatile DNA transformation system.Unlike most...similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle (a natural...pombe plasmids oftentimes utilize an ARS to aid in high transformation efficiency; however, this region ...antibiotic selection when working with yeast, due to high rates of spontaneously occurring resistant mutants...in the cell, thus allowing the yeast to maintain higher copy numbers. Additionally, improvements in antibiotic...

... antibody has arrived, hooray! Before you jump straight into the exciting new experiments that you’ve ... their ability to bind to molecular targets with high affinity and specificity. However, there are countless...validation in different applications. Here, I want to highlight the general suggestions given by the International...your bioinformatics skills (or your friendly neighborhood bioinformatician) to look at RNA expression ... tissues.  From this data, you see that there is high RNA expression in the retina and in parts of the...binding to something unexpected. This approach has high specificity, but you need to be aware of any possible...for immunocapture applications and is amenable to high-throughput analyses, however, it requires access...

...Nye in their lab coats handling glassware with brightly colored hazardous liquids. Working in a lab is...school, med school, industry) Other roles you might see Lab manager: managerial duties ex: order materials...makes them excited to do what they do. I would also highly recommend talking to other undergraduate researchers...choosing a good scientific mentor and choosing the right lab. Remember that your time is valuable! Don’t ...scientists are all just people. I’ve found that in the right environment and with supportive mentors, being open...was a bit of a foreign concept to me when I was in high school, but I’ve learned the value in finding mentors...broadening your experiences! These applications can be highly competitive, so asking for help from mentors and...

...by large DNA recognition sites, which makes them highly specific.  Jurassic Park by Michael Crichton was...Jurassic Park (1997, 2001, 2015, and 2018) were highly successful in the box office, indicating the insatiable...increase specificity. This modular approach made ZFNs highly appealing to researchers.  TALENs In 2010, transcription...travels. The movie sees the USS Enterprise crew fight against a genetically engineered superhuman Khan...cellular DNA repair machinery, which can lead to high frequency of indel formation in the genome and are...involves The Rock and his super giant albino gorilla fighting off a monstrous wolf and crocodile, that were ...Editing Trading Card seen in this blog post?Download high resolution versions here.  Many thanks to our guest...

...Addgene we've set out to fill this gap by both highlighting that researchers do learn MANY transferable ...the lab and by offering advice on areas where you might need some help. Today: Teamwork. My first experience...was I to dispute history? And boy did I make the right decision. If I’d spent my senior year isolating ...colleagues, I can also orchestrate the formation of high-functioning-but-temporary teams that have a specific...providing technical customer service. Team members are highly trained, and though there are necessary redundancies...skills - you just want the necessary skills. You might find that your next paper is going to require help... of more than one person. A selfish part of you might not want another person’s name on your next paper...

...clone out of 104 to 105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability... center of the sequence. Since lambda red has a higher recombination frequency when the lagging strand...strands. One of the two oligos will recombine with a higher efficiency than the other and you’ll find out empirically...frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing oligos that avoid activating...deletion of >20 nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase to generate insert ssDNA...these strains, expression of exo, beta and gam is tightly regulated by the endogenous phage promoter pL and... allows for a mobile recombineering system, but tight regulation of expression is required for a successful...