...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially... roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...healthy as can be to prepare them for the arduous process of generating viral particles. Limit tube transfer...

...various components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of ...drug reaction. This system would somewhat act like a 2-step verification. All approaches have their own limitations...Future Enhancements. Frontiers in drug discovery, 2, 952326. https://doi.org/10.3389/fddsv.2022.952326Joubert...approach cannot replicate the complexity of antigen processing events that take place within an antigen-presenting...

...non-specific proteins, and then elute YFP.  Option #2: Opto-Nanobodies (OptoNBs) purification You skip adding...blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled protein...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...with a flip of a light switch would improve this process. The team used nickel-coated agarose beads coated...

...research experiences to undergraduate students (1, 2), but educators face significant challenges in designing... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...Biology Education: A Call to Action, Washington, DC. 2. President's Council of Advisors on Science and Technology... controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to the C-termini...successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students first transform...

...typically, normal (bell curve) or skewed (see Fig. 2). Assumptions – characteristics of a data set; ...levels of variance among data.     Fig. 2: Examples of data distribution profiles.   Parametric... positive is much higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise ...statistical facets of reproducibility. Adv Physiol Educ. 40(2): 248-252 (2016). 0.1152/advan.00042.2016 Finch, W...Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389/fams.2016.00002  ...Bonferroni Correction and the Benjamini-Hochberg procedure are designed to reduce false positive rates and...

...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...transfection, transfection reagent, or transfection process) may have on the target cells. One transfection... the transfection reagents or the transfection process itself has any cytotoxic effects on the target ... optimization of the transfection reagents and process. It is important to note that optimal experimental...

...pH visible at the sample interface upon thaw (Fig. 2). Before thawing, all the samples were at the control...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...always looking for ways to improve our shipment processes. After reading a publication describing how short...

...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...scientists have been able to study many cellular processes, including mitosis, mitochondrial fission/fusion...insertions can only be detected by labor-intensive processes like PCR or by evaluating visual phenotypes. Improving...

...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking.... Protocol Pages Agarose cloning method procedure The procedure (illustrated in the YouTube video below...

... will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models reinforcement...spend most of its times in the opposite chamber) [2]. Laser activation is usually controlled automatically...two different ways 1) your hypothesis is wrong or 2) there is some problem with the technical implementation...PMID: 21716290. Pubmed Central PMCID: PMC3775282. 2. McDevitt, Ross A., et al. "Serotonergic versus nonserotonergic...neuronal activation and is a sufficient readout to proceed with your behavioral experiments. Comparison of...

... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...useful for the biotech industry, which is in the process of capitalizing on phage RBPs as diagnostic tools...DNA fragments with live host cells Now you can proceed with incubating this membrane with your cells of...

...   Figure 2: High-level overview of an indirect immunofluorescence procedure with key considerations... several steps outlined below and in Figure 2. For a deeper dive into the steps and techniques, see Im...recognize the species of your primary antibody and 2) is conjugated to a fluorophore that suits your experiment...primary antibodies is a time consuming and expensive process. Consider the vast number of possible primary antibodies...Performing an immunofluorescence experiment The process to perform IF can be broadly divided into the several...specific 3-dimensional epitopes. Different sample processing steps can alter epitopes in different ways, so...secondary antibody you use, so as long as the sample processing steps used in a non-fluorescent IHC/ICC assay...

... qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays...amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have a clear separation between...as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has ...one is needed. For these reasons, we are in the process of transitioning our titering method from qPCR ...information needed for your PCR experiment. The process to titrate AAV by ddPCR begins with diluting the...

...Quotes taken directly from participants (summarized in 2) touched upon many problems in this area: “ [The] ...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. McDowell, Gary S., et al. "Shaping the Future of ...A fair deal for PhD students and postdocs." Elife 2 (2013): e01139. Pubmed PMID: 24137543. Pubmed Central...biomedical research from its systemic flaws." Proceedings of the National Academy of Sciences 111.16 (2014...

...discussed them in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was...communication has helped Addgene move from completing 2-3 large projects a year, on average, to completing... academic lab or your workplace, is an ongoing process. It is never really done. But if you implement ...

...experiment using smFPs and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures...similar proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...central nervous system (CNS) orchestrates complex processes enabling organisms to control their movements ...tendency to brightly label somata but not neuronal processes. In contrast, smFPs can label fine neuronal structures...

...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...user-controlled illumination with violet light and (2) exposure to increased intracellular calcium. It has...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded...to your parameters.   Kinetics How fast is the process you would like to monitor? Neurons, for example...

... as muscular dystrophies, heart disease, and type 2 diabetes, across four different types of human cells...(#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...array  Although CRISPR-Cas12a, with abilities to process and release its own gRNAs from a single array, ...efficient base editing and prime editing, is unable to process its own array but is the key component to enable...minimal size. To do so, they harnessed the tRNA processing mechanism, which is conserved in all living organisms...individual gRNAs are released by endogenous tRNA processing machineries, specifically, RNaseZ and RNaseP....additional promoter, which is followed by tRNA processing to release individual gRNAs for gene editing....

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...optogenetics to study physiological and behavioral processes (Abdallah et al., 2018; Deubner et al., 2019)....induces reanimation from general anesthesia." Proceedings of the National Academy of Sciences 113.45 (2016...