...pooled libraries have allowed scientists to easily perform genome-wide screens to effectively and efficiently...human genome-wide CRISPRko (knock out), CRISPRi (interference), and CRISRPa (activation) libraries that do...that improved sgRNA activity and thus library performance. The Root and Doench lab’s optimized genome-wide...CRISPRi, and CRISPRa libraries have been shown to outperform other popular CRISPR libraries (see below). Let...colon cancer) cells and assessed the libraries performance via the depletion of a gold standard set of genes...developed sgRNA-level metrics to assess library performance in viability screens. Based on these metrics ...sgRNAs, Brunello even with only a single sgRNA outperformed another CRISPR library with 6 sgRNAS, highlighting...

...protocols and performing rigorous quality control (QC). The specific QC experiments performed varies for ...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...and stored at -80 °C. Titer Titering is either performed by Addgene or by the University of Pennsylvania...Pennsylvania Vector Core. In general, titering is performed by the facility that produced the viral vector lot....Pennsylvania Vector Core is (as of April 2016) also performed by ddPCR.  Quality Control Addgene ensures high.... To learn which specific QC experiments were performed on your lot, please contact us. Full sequencing...the Viral Genome Next-generation sequencing is performed on viral genomes isolated from the final AAV preparation...

...scaling. Prokaryotes have long utilized CRISPR as a powerful defensive strategy against viral invaders, and...

...lentiviral CRISPR libraries (both human and mouse) for performing genome-wide knockout screens. The libraries are...

...is, an antibody shown to perform well in one set of conditions may not perform as well in other conditions...approach is RNA interference (RNAi). The most common method is transfection of short interfering RNA (siRNA...assumed (Ayoubi et al., 2023). Additionally, their performance depends on the specific protocol used and the...For example, we have found that some antibodies perform well in a PFA-saponin fixation and permeabilization...population (Figure 1), further optimization can be performed to both maximize the separation and reduce any... is possible to design experiments where the performance of the antibody can be assessed through its ability...different conjugates. Summary Flow cytometry is a powerful tool to delineate cell populations and to study...

...your antibody performs as expected in one application does not mean that it will perform as expected in...doesn’t necessarily mean that you yourself have to perform extensive validation experiments for every antibody...data, you may need to roll up your sleeves and perform some extra validation experiments yourself.  Goal...researchers can use to show that their antibody performs as expected. One of the benefits of these pillars...levels tend to correspond to protein levels, so you perform immunohistochemistry with your antibody on these... the epitope recognized by your antibody. You perform immunocytochemistry on these mutant cells as well...caveat is that different antibodies may or may not perform well under the same conditions. Example: You want...

...homemade competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications will require... pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr protocol prior to...adopted by any laboratory. All procedures are performed using the same three step protocol:  Fragment...efficiency of the strain: when asking the cell to perform multiple recombination events, all linear fragments... design (Figure 2), IVA cloning can be used to perform any plasmid modification, from insertions, deletions... reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride the old codon, amplifying...primers can be used in combination to simultaneously perform modifications, simply by adding all the necessary...

...contains five plasmids and one bacterial strain for performing a bacterial two-hybrid (B2H) screen of modularly...

... line to perform downstream characterization of, isolating 2 or 3 distinct clones to perform key experiments...CRISPR-Cas system localizes to unintended sites and performs its programmed function there, this is an off-...off-target effects, but it’s important to not let ‘perfection’ get in the way of ‘good enough’. Predicting ...would confound your experimental results and 2) perform targeted off-target measurements at those sites... to quantify off-target effects entirely is to perform whole genome sequencing. While this may be unnecessary... The con to these approaches is that NHEJ must perform the repair to allow for detection, not Homologous...targets as well as clonal differences. If you are performing CRISPR library screens, the same principle holds...

...contrast technique in modern biology (1). When performed correctly, fluorescence microscopy provides a ...contrast image in which a bright signal overlays a perfectly black background. In addition to this, the use...confocal microscopes use parallelization to enhance performance. In spinning disk microscopy, a metal disk containing...coverslip at an angle such that when it reaches the interface between the glass coverslip and a sample in aqueous...within a few hundred nanometers of the glass/water interface. Super resolution microscopy Super Resolution ...means that the images will appear sharp without interference from blurry out-of-focus light. The most difficult...be needed. Most commonly these experiments are performed on a fluorescent widefield microscope. Here, excitation...

...Mikko Taipale 12563 PCD FL0X Firefly Insertion of imperfect miRNA sequence in the 3' UTR Carl Novina 33154...

...extracellular fluid into the perfused liquid. The probe then collects the perfusion liquid for quantitation...fluid (Darvesh et al., 2011). This technique is performed with a probe surrounded by a semipermeable membrane... quantitation is desired (Figure 1). The probe perfuses liquid, which causes the passive diffusion of ...The advantage of this method is that it can be performed in a live awake animal by surgical implantation...into a specific brain region. While the method is powerful and offers immediate measurements in a live animal... GRABDA sensor here! Overall these sensors are powerful new developments in the detection and measurement...

...predict how fluorophores perform in ‘real experiments’, it is advisable to perform time-lapse imaging of ...To determine photostability, an experiment is performed that measures fluorescence intensity over time...Therefore, photostability measurements can be performed with soluble fluorescent proteins or localized...photostability measurements reported in the literature are performed in different ways. We describe a number of issues...fluorescent protein. Photostability measurements can be performed on purified fluorescent proteins. To avoid diffusion...changes in excitation power, it is important to perform the experiments on a single day on a stable system...

...donated MiSeq.  For Complete Plasmid Sequencing we perform a 2x251 run on our MiSeq that takes about two days...involves a significant amount of QC. First, we perform Picogreen quantification on each and every isolated...samples for Viral Genome Sequencing (VGS). QC is performed throughout the library preparation process and...Aligning the sequence The first step we take when performing quality control of newly deposited plasmids is...backbone sequence. We’re not necessarily expecting a perfect match - we will often find a few mismatches in ... reads to the correct sample. The PCR is then performed, optimized as needed, and cleaned up using a standard...to remove any template DNA and primers.  After performing extensive QC on that sample, we’ll then load ...

...for endogenous gene activation Optogenetics is a powerful tool that utilizes light to control and monitor... affinity for SspB1. In order to create a more powerful LID, the authors recently used computational protein...pathways. These LIDs are generalizable, versatile and powerful tools which allow you to easily and reversibly...elements in a single lentiviral vector provides a powerful tool to modify mammalian cells. Multicolor CRISPR...and broad host-tropism. These tools open up many powerful avenues through which one can examine molecular...all-optical electrophysiology is designing the perfect voltage indicator and optogenetic actuator pair...expression via western blot, to purify your POI, to perform pull down experiments like co-IPs and ChIP, and...

...adjacent areas impacted the experiments we wanted to perform. It required a great deal of time and effort to... reformulating our AAV construct to optimize performance in our system and yet none of that data was published...packaging viral particles (6), can influence vector performance. Open science initiatives, like Addgene’s Data... to help scientists understand how our vectors perform in various experimental settings. It is a curated...low-magnification images demonstrating vector performance) will be assigned citable DOIs through DataCite... broadening the knowledge base of AAV vector performance in various brain regions and cell types. Power...potentially available to evaluate AAV vector performance. Yet, most of the technical data needed to replicate...

...applications. An antibody that performs well in immunohistochemistry may not perform well in a western blot. ... than mammalian cells, but mammalian cells can perform human-like post-translational modifications that... If that’s the case, a buffer exchange can be performed using a desalting column or an ultrafiltration...ones, consider increasing the number of washes performed during your harvest. Validation Recombinant antibodies... bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin...characterization & rheology, Current Opinion in Colloid & Interface Science, Volume 19, Issue 5, 2014, Pages 438-449...

... with new techniques rapidly being developed. Performing a CRISPR experiment requires delivery of, at ...your genomic site of interest. This is commonly performed by transfecting cells with a plasmid, such as ...Cas9-gRNA ribonucleoproteins  An advantage of performing CRISPR experiments using RNPs is the variety ... RNP. This advantage has allowed scientists to perform in vitro and in vivo CRISPR studies in experimental...PEG-mediated methods, while technically less difficult to perform, often show low efficiency, while biolistic bombardment...plasmid based systems, most notably the ability to perform CRISPR studies in cells that are difficult to transfect...these have been acquired, genomic editing can be performed rapidly and, in many cases, with reduced chance...

...with purified enzymes, it takes advantage of the powerful recombination abilities of yeast. Similar to Gibson...than 10kb. Another advantage is the ability to perform oligonucleotide stitching, in which pieces of DNA...

...dTomato) that promote interactions between the FPs interfere with native interactions, preventing proper localization...cell-type dependent. Therefore, it is good practice to perform this assay with your chosen FP in your cell type...between studies is substantial (e.g, the poor performance of mRuby2 is detected by Constantini et al (2015...not by Cranfill et al (2016)). Moreover, good performance in the OSER assay is not a guarantee that using...relevant for your research. This will reveal the performance of fluorescent proteins in your biological system...resource with tools and data that indicate the performance of (a set of) fluorescent proteins under specific...