...PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via a novel capsid ELISA...report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing purposes...mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. Our titer...of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID: 10455443. ...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material." Human gene therapy 21.10...the other to guess a category by listing off things that fall into that category? Okay, let’s play. I’...’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot ...

...Part 2: Building Community  My editor’s choice pick for this month is “Grad School Advice Part 2: Building...Options for CRISPR Genotyping Grad School Advice Part 2: Building Community Engineering the Plant Genome Using...fantastic posts in October. Click on the links below to catch up on ways to use CRISPR in plants, to learn techniques...Writing 5 Reasons to Use Reddit for Science Communication Changing Labor Laws Bring Increased Postdoc ...

...below. Types of Voltage Indicators GEVIs can be divided up structurally into 2 main groups: voltage sensitive...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically...Voltage indicators are voltmeters for cells! In this blog we will review how these indicators work, their...fluorescence or voltage indicators.   Viral Vectors and Voltage Indicators As previously mentioned...Enhanced genetically encoded voltage indicators advance their applications in neuroscience. Curr Opin Biomed...Cellular communication and function depend on potential energy. Cellular activities like action potentials...their general applications, and why viruses are essential to their use as biological tools. Why Use Voltage...

... needing to work with a transfectable cell line. 2) They require larger expression vectors which are ...are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method... Cpf1 expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from...Biotechnology 35.1 (2016): 31-34. Pubmed PMID: 27918548. 2. Zetsche, B., Gootenberg, J., Abudayyeh, O., Slaymaker...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID... a simple and effective method with multiple applications (in vitro, in vivo, transfection, viral transduction...Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background with Our Guide Pages...

...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...run in triplicate.  The nice thing about qPCR is that since it measures relative quantification - that... not need to be run in triplicates. ddPCR is an extremely accurate application, even in areas of the genome...Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications Resources on Addgene.org...blog post, we're going to focus on PCR-based applications (qPCR and ddPCR.) qPCR Quantitative/real-time...range of RQ values as noted above.   In both applications, results can be affected by DNA quality and ...

...fly, E. coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries...targeting a specific gene within the genome (see Fig. 2). Individual gRNAs are designed in silico using publicly...CRISPR libraries is a multi-step process (see Fig. 2). In most cases, CRISPR libraries are provided at ... is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately...uncontrolled cell growth, such as cancer. Figure 2: The CRISPR library must be amplified (A) to be used... unique compared to the rest of the genome and located immediately 5’ to a protospacer adjacent motif ...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral...

...Topic) 0:00 - 2:00 : Introduction to the podcast from producers Tyler Ford and Eddy Page. 2:00 - 3:55 : ...MD PhD process and explains why he loved the first 2 years of medical school. 7:05 - 10:55 : Alex's focus...materials (check out our new Viral Service), and educational outreach. Many thanks to Alex Chavez for taking...

.... To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm ... of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561 ... stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The excitation...this example, I will describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab...efficiency (eGFP expression).     The first item in Table 2 is a standard and should always be included—the live...from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it...case the CD45 expression turns out to be weak. Table 2: A simple flow panel to detect live, transduced, and...

...organs. Restraint: scruffing Location: peritoneal cavity Gauge: 25–27 Volume: < 2–3 ml Skill required: intermediate... or mechanical restraint Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill...Intraperitoneal Scruffing Peritoneal cavity 25–27 < 2–3 mL Intermediate Intramuscular Mechanical restrainer...Back or hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia...Addgene blog Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms Additional resources .... It sounds complicated but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge... Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge: 27–30 Volume...

...help in multimer identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed... I hope these tips demonstrate that plasmid verification is not just necessary but also an easy process.... Please vist Addgene's resource for plasmid verification to find additional tips and detailed protocols...

... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ...functionality can be gained by understanding the location and transport of the protein within a living cell...to observe membrane trafficking events in vivo. Locate your protein and see who it is hanging out with...

...neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from the archaebacteria...Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proceedings...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

... Figure 2 that determining where to place your gate based on the dot plot would be complicated.  ...Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify and ...low expression, and everything in between (Figure 2). These smears tend to occur when you are looking ... Figure 2: (A) a smeary dot plot showing a range of antibody readouts. (B) A gate on the smeary dot...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...

...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...genetic underpinnings of these processes has been complicated by the lack of precise genetic tools for modulating...recent advancements and offer commentary on their application to neuroscience research. What is CRISPR-based...accessible to neuroscientists, not all neuroscience applications require such optimized tools. This is elegantly...necessitates such molecular gymnastics for sensitive applications such as CRISPR screening. The plasmids used ...used by the Qi Lab for this particular application can be found here.  Why CRISPRa? Although cDNA overexpression...

...improving editing accuracy by preventing re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore...also takes into account that HDR is rare, usually 2-5% of the edited cells in our hands, these combined...screen a few hundred clones to find the right one Step 2 – Edit the cells a second time using a repair template...Additional CRISPR Resources on Addgene's CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though... wisely (and let them cut close).   Figure 2: Relationship between cut site to mutation distance...PMID: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering...many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous...

...question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1,...however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The protospacer flanking sequence...PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct RNase... and functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385...use, functional in most species, and has many applications. That said, SpCas9 is not the only game in town... makes this molecule unique, and the various applications of Cas13a. The origins of Cas13a: An RNA cleaving...53 potential candidate genes that fell into 3 categories based on the architecture of the CRISPR protein...

...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the different...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization through...Glutamate plays a variety of roles in synaptic communication and can trigger other forms of neuronal signaling...SF-Venus-iGluSnFR). iGluSnFR3: Improved Glutamate Indicators for Synaptic Imaging However, the first two generations... 30377363 Aggarwal, Abhi, et al. . “Glutamate indicators with improved activation kinetics and localiation...

...observed one complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where...Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received plasmid requests from ...observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID-19 ...there were zero plasmid requests. Afterwards, from 2/24/20–3/15/20 (the supposed end of lockdown), request...However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown in China...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...14%). Spikes in plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests...

...Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain...resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...machinery targeting 1) the given resistance gene and 2) a second lytic phage. Bacteria that receive this ...bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics...237163. Find plasmids from this paper at Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such...