...using a high-speed ultracentrifuge. During the spin, the virions will form a band due to their high density...nationwide where you can obtain excellent quality, high titer rAAV (including Addgene!), but you can also...terminal repeats (ITR). You should always have a high-quality plasmid standard and rAAV reference material...Zen, Z., Espinoza, Y., Bleu, T., Sommer, J. M., & Wright, J. F. (2004). Infectious titer assay for adeno-associated...

... backbone might be more prone to recombination, while a library with a large backbone might be more difficult...to ensure the highest possible transformation efficiency. Using Stbl4 or other ultra-high efficiency cells...specifically calculated based on the size of and weight of your library plasmids. As long as you achieve...

..., and so on and so forth. This also serves as a light technical control - an RQ value of 1.5 means you...quencher to quantify specific amplicons.  Sybr has high background noise, due to the large amount of dsDNA...DNA and the length of the amplicon and it has a higher rate of false positives (Colborn, 2008; Harridan...number of factors will determine which approach is right for your experiment. You’ll have to consider:  ...analysis of copy number variants based on real-time LightCycler PCR. Curr Protoc Hum Genet. 2014;80:7.21.1-7.21...

...cells to grow and divide only at the right times and in the right places. Checks and balances: protecting...proteins naturally lack areas that are accessible, high-affinity binding targets for potential therapeutic...therapeutic agents. Nevertheless, a glimmer of light remains for this elusive target. Recent improvements in ...results and customize the small molecules for a higher affinity fit. Kevan Shokat’s group at the University...

...now, 2 decades later, is it finally back in the limelight. On December 19, 2017 the FDA approved Luxturna... brought all these key elements together at the right time and place. But it is the hard work and persistence...immunogenicity is critical when systemic delivery of high doses of vectors are needed. Additionally, AAVs ...ultra-rare ocular diseases are being benched due to the high cost of clinical development. As a result, Luk Vandenberghe...8. Trapani I and Auricchio A., 2018. Seeing the Light after 25 Years of Retinal Gene Therapy. Trends Mol...

...project she needs one of you to lead. At first you might think, “I’m not a leader,” because you don’t see...you. How is this new opportunity much different? Might you already have leadership skills that are just...and develop your nascent leadership skills. You might just find that you’re more of a leader than you ...External collaborators For further advice on highlighting your transferable skills in that big job interview...

...ancestry groups rather than self-reported race, it highlights the myriad issues researchers need to reflect...associated dependencies across ancestry groups (right).    Seeking an explanation  First, they looked...guides from being designed that target regions with high genomic variability.   Moving forward It took a ...Stephanie M. Fullerton et al. Getting genetic ancestry right for science and society." Science 376, (2022): 250...

...excluding Starbucks. Taking a nerdy perspective, you might denote your search as follows: (my_house + coffee...math illustrated. Intersect (left) and set minus (right). Enhancers are regions of DNA that demonstrate...enhancers, check out the Appendix. Conclusion: Shedding light on dark matter Precise knowledge of which stretches... courtesy of beyondthedish (left) and biobabel (right). People commonly think of DNA as a linear polymer...meters long. You twist and roll the string into a tight ball. Now the two ends of the string, instead of...

... may be just the method needed to jumpstart the fight against antibiotic resistance. Advantages of CRISPR... despite this resistance gene being encoded by a high-copy plasmid. In the case of the genomically encoded...distinct beta-lactam resistance genes, again with very high specificity. This double phage system both favors...bacteria. In mixed populations, their phages were highly specific for certain bacteria, just like the CRISPR...new strategy can help us win back ground in the fight against bacterial superbugs. References 1. Citorik...

... species, Zetsche et al. hypothesized that Cpf1 might represent a functional CRISPR nuclease that could... cleaves relatively far away from the PAM, NHEJ might retain the PAM site. Therefore, if HDR does not ...possibly mediate HDR. This “second chance” mechanism might improve the frequency of desired HDR edits, but ...editing efficiency for the Cpf1 orthologs was only slightly lower than that of the widely used SpCas9 and ...that AsCpf1’s off-target rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1...

...has drastically increased, it would have been a tight squeeze for all of us to fit in Joanne’s back yard...discovered my hidden talent in Ladder Ball... there might be a possibility it was just beginner’s luck, but... (only if it’s cool with your legal department) right in your breakroom. 5. Throw some physical activity...you have a very intricate itinerary, there is a higher likelihood that something will go awry. Don’t plan... My grandma used the old adage “many hands make light work” all the time. She was a wise lady for many...

...shielded from quenching by aqueous solvent. This tightly-packed structure explains the importance of the...permutations within each color also exist with only slight variations separating them.  Please note that many... Improves folding at 37C, reduces aggregation at high concentrations T203Y Red-shifts the wavelength...biosensors here. Optogenetics: Scientists can use light to detect, measure, and control molecular signals... blog, you should! He's got great articles and insight on the newest tools and techniques in fluorescent...

... This powerful negative selection system ensures high editing efficiency; the only non-edited cells to...The simpler system of pCRISPomyces-2 displays a higher editing efficiency, perhaps due to its condensed...Streptococcus thermophilus, Cas9sth1 as a robust and highly active enzyme for transcriptional silencing Mycobacterium...efficiency of knockdown and developed new, more tightly controlled, Tet responsive promoters to avoid leaky...dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that further improves.../10.1038/nbt.3011 Cobb RE, Wang Y, Zhao H (2014) High-Efficiency Multiplex Genome Editing of Streptomyces...

...found that the accuracy of ICE analysis results was highly comparable (within r2=0.96) to that of NGS data...coefficient (r) is also computed and reported.  The higher the R2 value, the more confident you can be in ...the Download Analysis Data button on the bottom right of the analysis screen. ICE analysis details Contributions...genomes that contain the indel. The discordance plot (right panel) shows the level of disagreement between the...that continues after the cut site, representing a high level of sequence difference. Traces The Traces...

...SDS-PAGE...Electron microscopy… Any guesses? That's right... ways to titer AAV! The point is there are many...linear region of the standard curve. In addition, high and low-concentration samples can also behave differently... reference sample of known titer, which is at a higher titer than the AAV RSM. By using these samples ...) AAV titers are approximations and they depend highly on the experimental conditions used, titer values...PMID: 10455443. 3. Hindson, Benjamin J., et al. "High-throughput droplet digital PCR system for absolute...

... develop and optimize the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK... the promiscuous cleavage and degradation of neighboring ssRNA and ssDNA by Cas13 and Cas12a, respectively... of CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (...SHERLOCKv2 provides a method to detect nucleic acids with high sensitivity and specificity without compromising...the ability to rapidly detect nucleic acids with high selectivity and sensitivity from patient samples...

...technology has become very popular as a rapid and highly efficient way to move DNA sequences into multiple...two new recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL ...’ll walk through an example experiment where we might use Gateway cloning to generate our desired constructs...another, the inserted DNA fragment stays in frame. High efficiency The positive (antibiotic) and negative...M. (1992). A Genetic Switch: Phage (Lambda) and Higher Organisms (Cambridge, MA: Cell Press). Additional...

...three scenarios, but a short treatment regiment might wipe out bacteria that are completely susceptible...concentrations of a drug, but will be killed by a higher concentration, or sometimes more exposure time ... corresponds to the level of susceptibility you might expect if a certain antibiotic were chosen for treatment...survive for millennia. What we can do, however, is fight smarter, not harder. We can utilize tactics like...antimicrobials to learn how scientists are using CRISPR to fight bacteria. From a societal standpoint, we can all...

...Department of Labor’s general guide to the effect on higher education, you can see that those in teaching roles...lot of us, frankly quite surprising. Consortia of higher education institutions submitted letters to the...exemption. Discussions about the drastic effects on higher ed had been seized upon by opponents of the ruling...convincing as the recommendations for postdocs to be on higher salaries (which have been made by multiple organizations...recommendations that postdoc salaries be raised to $50,000 - higher than the level they will be raised to on Dec 1st...

...you can see some of the stats from the AMA to the right - the Reddit community is active, engaged, and has...resources at her disposal, she was writing all day and straight on through her designated two hours for answering... to other scientists who will find it useful, I highly recommend doing an AMA! Before I turn it over to... Kudos to the moderators for keeping the quality high and thus enabling a productive discourse. They even...to resources. I’ve said this before, but I also highly recommend taking every opportunity you get to speak...