...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...

... 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...

... 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...

...   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...

...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 ...

...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...

...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...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative...randomized reorganization process, creating a unique binding site. This process, known as V(D)J recombination...delaying a process through sequential rounds of validation. Validation of an antibody is a process separate...

... 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...

...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126...are essential tools for visualizing biological processes in vivo. However, mScarlet’s slow maturation rate...like the C. elegans embryo, where developmental processes happen within a few hours. In order to use the...addition to the toolkit for visualizing cellular processes in C. elegans: mScarlet3 for applications needing...

...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...Organisms, Part 1 Five Popular Model Organisms, Part 2 ...’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project. Gymnotiformes and Mormyridae...evolution in immunology, neurobiology, and more. Prochlorococcus (Penny Chisholm) This cyanobacterium is the...need to grow to get a recombinant because this process is expensive. They are using a cool fluorescent...

... Figure 2: Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming ...ironing board, giving it the perfect height (Fig. 2). We recorded video using a simple, external video...doesn’t, and trying all over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn...gear, and walking everyone through the recording process was going to be time consuming and difficult. Finding...in the future, but we were finally refining the process down to a place I could be happy with it. Cycle...

... environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in plasmid...debris”; and then clean-up the plasmid DNA (Figure 2). For most kits, the steps for lysing cells and removing...together — no mixing and matching!   Figure 2: Plasmid isolation workflows are similar across kits...that can arise during the plasmid purification process. One is the possibility of damaging the plasmid...carrying over contaminants from the plasmid prep process into the final plasmid eluate. Possible contaminants...contaminants can sneak in from almost any step of the process and include things like genomic DNA, RNA, proteins...

...delicate axonal and dendritic processes not previously visible with Brainbow-1 and -2. The general structure ...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...

...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations...used method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...Statistically defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. ... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...Getting your RQ value isn’t the end of your analysis process. You’ll still need to do statistical analysis to...Gene Expression Analysis During iPS Reprogramming Process. Sci Rep 8, 8716 (2018). https://doi.org/10.1038...

...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates...vectors Learn about Addgene AAV Quality Control Process Resources on Addgene.org Find ready-to-use AAV...