...applications (qPCR and ddPCR.) qPCR Quantitative/real-time PCR (qPCR) has been used for copy number assays ...or negative for each reaction, math happens (this time, Poisson statistics), and the readout gives an absolute...Gommersall L, Arya N, Patel HR. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn. 2005 Mar...Quantitative analysis of copy number variants based on real-time LightCycler PCR. Curr Protoc Hum Genet. 2014;80:...hg0721s80 Colborn JM, Byrd BD, Koita OA, Krogstad DJ. Estimation of copy number using SYBR Green: confounding ...Na-Bangchang K. SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination...determined by SYBR-Green-based quantitative real-time PCR. J Microbiol Methods. 2006 Jun;65(3):476-87....

...on the Addgene Blog Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User) The Challenges of Cell...colonies of antibiotic-resistant cells; during the time required to grease, place and fill a dozen cloning...dilution cloning into 96-well plates, is extremely time- and resource-consumptive, since only wells with...well-spaced clones. For rapidly growing cells we sometimes use TPP peelaway T-75 flasks to provide a greater... in outgrowth. Depending on the cell type, we sometimes add a drop of serum to each well in the 48-well... the University of Maryland Medical School in Baltimore. She is particularly interested in signaling peptide...

...languages? That's trickier. Sometimes it's the developers. But, it turns out over time that the most successful...use plasmids, they are trying to do one thing at a time. What the BioBricks Foundation is seeking to accomplish... up, and slam that onto a mass spec. That's very time intensive and expensive. There's the promise that...seen the development of enough sensor domains. Ultimately, we'll need a couple thousand or so sensor domains...bioeconomy is already pretty substantial. It's estimated that genetic engineering as applied to products...want is 93 percent. All of a sudden, for the first time, you've got a collection of reusable, off-the-shelf...

...some genetic diseases. I didn’t know it at the time, but gene therapy had fallen - plummeted really ... organ that can be monitored non-invasively over time. Importantly, it is an immune privileged compartment...destructive inflammatory immune response. At the time, adenoviral vectors were the most commonly used ...cells - AAV - were becoming available. At the same time advances in human genetics led to the identification...brought all these key elements together at the right time and place. But it is the hard work and persistence...deliver via AAV.  Dual vectors strategies are being optimized to overcome this limitation, with limited success...designing expression cassettes to regulate and optimize transgene levels, and large scale manufacturing...

... you may need to run the experiment a few times and optimize your standard curve accordingly.    ...later.       Standard curves must be included every time the assay is run, ideally with freshly prepared ... including the user, equipment, assay incubation time, etc., which can vary even when the same protocol...curve? A poor standard curve leads to over or underestimation of an unknown sample’s concentration. To determine...sample does not fall within this range, it may be time to reconsider your graphing program. Summary Few...instruments and specific assays you will be using ahead of time to see what factors may impact the dynamic range...

...perform. It required a great deal of time and effort to test and optimize our vector - overall, I spent two...within the same brain region or nucleus. Just optimizing AAV expression can take months (or longer), yet...testing and reformulating our AAV construct to optimize performance in our system and yet none of that...to see more negative data (Figure 2.) At the same time, when scientists who support open science, but do... C., Garner, K. M., & Adan, R. A. H. (2010). Optimization of Adeno-Associated Viral Vector-Mediated Gene...Researcher Perspectives on Data Sharing in Deep Brain Stimulation. Frontiers in Human Neuroscience, 14(December...

...slog through unappealing work in your precious free time.  And don’t be afraid to think outside of the box...project, I can guarantee you that either resource or time restrictions (or both!) will be a major limiting...understand what kind of resources - including your time - will be required to produce an outcome you’re ...proficiency.  What kind of content are you sharing?  Sometimes your subject matter will drive the kinds of communications...presentations, etc… Low (except for space) Real-time audience engagement and response. High connection...resource geographic location) Opportunity for real time engagement while allowing for recording/future access... speed internet access. High probability of real-time technical difficulties Exhibits Medium to high...

...can be shortened since the plated cells will have time to produce beta-lactamase prior to division, instead...effects of the antibiotic. This buys you a little more time, since bacteria won’t divide immediately after it...beta-lactamase. Pros: Widely available, cost-effective, timesaving for transformations. Cons: Prone to satellite... Pros: Very stable, interchangeable with Amp, timesaving for transformations. Con: Expensive.   Kanamycin...resistance Choosing the best antibiotic resistance ultimately comes down to your applications and needs. The...selecting!   Antibiotic Resistance Stability Time Saving?  Works for multiple organisms? Compatible...storage temperature and pH on the stability of antimicrobial agents in MIC trays. Journal of Clinical Microbiology...

...for some time now. When titrating AAV by qPCR, viral DNA is amplified and monitored in real time. When the...s. Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has been shown to.... But what if we could repeat this assay 10,000 times to ensure that our titers were as accurate as possible... or a reference, meaning saving on reagents (and time)! AAV titering using droplet digital PCR Droplet... that could potentially lead to contamination. Optimize your PCR For a good starting place on what PCR...

...neuroscience, including transgenic mice, neuron-optimized viral vectors, and high-throughput screening approaches...Figure 1b), and sgRNAs can be found here. Neuron-optimized viral vectors Alternatively, Cas9 activators can...recently by the Day Lab, who developed neuron-optimized lentiviral vectors for in vitro and in vivo CRISPRa...with BioRender.com. The Day Lab used their optimized vectors to highlight a key advantage of CRISPRa... to neuronal physiology. The Day Lab’s neuron-optimized lentiviral vectors can be found here. High-throughput...not all neuroscience applications require such optimized tools. This is elegantly demonstrated by the Qi...PMC6214761. Savell, Katherine E., et al. "A neuron-optimized CRISPR/dCas9 activation system for robust and ...

...your screen result may or may not actually help. Ultimately, if you cannot describe the likely outcome with...grant. Either way, think realistically about the time and resources required to feasibly accomplish the... address the aims in more depth in the remaining time. The typical chalk talk structure is: What is the... details. Make the most of your limited speaking time. Beyond the information you seek to convey in your...feedback. It’s also helpful to practice at least a few times with people that are outside your lab and outside...or while the committee is arriving. Spend as much time as possible facing the committee and not the whiteboard...secretory proteins in the Endoplasmic Reticulum, optimization of fluorescent proteins,  and live cell imaging...

...us, such as finding time to read all of these articles. Most of us hardly have time to keep up with all... our own topic. Only on occasion do we then have time to read about research from other disciplines, or... of interdisciplinary research while at the same time increasing interdisciplinary citations in their ...videos of 3 to 5 minutes provide more than enough time for researchers to describe their science, and for... Rely on Circadian Rhythm and Melatonin for the Timing of Nocturnal Courtship Vocalization. The video ...youtu.be/EHHhiawIdWM Saving salmon, one embryo at a time – A video by LeeAnne Thayer, a Ph.D. candidate in...

... gene you’d like to manipulate in mind. Now it’s time to consider where you would like to attack this ..., 2014. Not all gRNA’s are effective. At the time of writing many of the specifics surrounding why... remain unknown, but fret not! The key to saving time and energy during your in vivo work is to screen...similar plasmids for gRNA expression), one gRNA at a time. After 48 hours of incubation, you assess the level...technique, you can assess several sgRNA’s at the same time, and choose the most effective ones to increase ...validated your gRNAs for use in mammalian cells, it’s time to use them in mouse embryos. When researchers first...mouse embryos to develop. Once they are born, it’s time to screen them for your desired edit. Good news!...

..., in actuality, many find using lentivirus to be time consuming, difficult, and lacking in reproducibility...enough to promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the cell cycle...efficiencies. Cultures should only be passaged 20-30 times before discarding and thawing a fresh vial. In many...prefer to collect and pool the harvests from multiple time points while others opt for a single harvest after...lentivirus are determined by the envelop protein. Many times lentiviral vectors are engineered with non-native...such variables as cell density, volume, incubation time, and MOI can all impact the success of your lentiviral... S., Guo, S., Xu, J., Kappes, J.C., Hel, Z. “Optimization of the transductional efficiency of lentiviral...

...is that access of this data is to be made at the time of manuscript submission. The rationale includes...these were those from researchers who wanted more time to mine their own data, with some smaller labs feeling... originator was oblivious to, no matter how much time they had. It could be argued that data generators...having exclusive access to data and enjoy reasonable time to make sense of it, with their decision to publish... specific (expensive) software that evolves over time such that prior formats are no longer supported/... especially for young researchers who may be intimidated by having to contact more senior colleagues. ... kinases implicated in human diseases. His spare time is spent trying to raise the alpha isoform of GSK...

...this time, Dr. Koeris and Professor Timothy Lu (a graduate student in the Collins' lab at the time) got.... As you'll hear Dr. Koeris explain however, the time was not right for the development of phage-based...listening! Download the podcast here! Podcast breakdown (Time : Topic) 0:00 - 1:25 : Introduction 1:25 - 6:19 ...   We'd like to thank Dr. Koeris for taking the time to talk to us and taking up a position on the Addgene...

...interest as possible, some optimization will be necessary. To begin, one should optimize protein production using...phosphoserine directly into recombinant proteins (5). Optimization of this system defined three components required...phosphoserine, the Sep-tRNA synthetase, and EF-Sep – an optimized elongation factor that accepts Sep-charged tRNAs... have worked most extensively worked with and optimized Sep-OTSλ and can recommend its use (6).  2. C321...that can be used as a positive control during optimization experiments. This version of GFP will contain...purification strategies; expression should be optimized for parameters such as induction temperature and...

...continued this tradition. We now have over 30 full-time employees, so we created one long lunch table and...bring in baked goods and we have a few unique lunchtime traditions that might be fun to incorporate into... take a break from their everyday tasks and make time for special projects? We borrowed an idea from the... success at accomplishing “we-should-really-make-time-for-that” type projects. Cross-team “Random Lunches...between labs. Scientists are often willing to make time for free food, and these lunches would provide a... as important to take a break from work to share time with the people around us. It makes the quality ...

...develop an optimized photoactivatable CRISPR gene activation system, but settled on a different optimal fusion...to use light to externally control the location, timing, and reversibility of the genome editing process...researchers tested a variety of combinations to optimize both fusion proteins. The best performing combination...fusion protein combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the... prevent DNA binding in the dark. Upon light stimulation, the pdDronpa moieties move apart, allowing DNA...LOV2 has a C-terminal helix that unfolds when stimulated by blue light. When fused to the catalytic site...chromatin architecture, it is useful to regulate the time in which dCas9 binding occurs. Bubeck et al. expressed...

...nucleome, the organization of the nucleus in space and time, and to understanding how nuclear organization varies... change in response to the cell cycle or other stimuli.   To create a colorful Cas9 toolbox, Ma et al....study is the method’s sensitivity. The authors estimate that a minimum of 150-200 fluorescent protein ...phototoxicity concerns and extending the potential imaging time for this technique. The NIH Common Fund has made...epigenetics and cellular reactions to external stimuli. It could also have major applications to cancer...imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Chen B, Gilbert LA, Cimini ...