...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent... PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...importantly, most Cas9 orthologs are less well characterized than the conventionally used SpCas9 (derived...

...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...production of tag-free proteins for further characterization and analysis. In a rapid response to the COVID...Versatile High-Throughput Cloning System for Characterization of Enzyme Variants. In: Methods in Molecular...

... 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...the chosen brain circuit, while a nose-poke at a separate hole (inactive hole) does not. If activation of... help to refine and optimize your stimulation parameters as well. Inhibition of neuronal activation is...contextual fear memories [5].  In a modified behavioral paradigm, the authors found that precise but not prolonged...

...   AAV2 Heparan sulfate proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...suggest these serotypes as a good place to start. Table 2: AAV tissue tropism for mice in vivo. To target... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9),...Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...Formally, pseudotypes are described with two numbers separated by a slash: the first number refers to the genome...but some AAV9 tools are available as in-stock preparations. AAV retrograde (AAVrg, also known as AAV2-retro...

...use 2 different secondaries for your antibody pair and visualize both targets in parallel (Fig. 2). For...successfully visualize both proteins.     Figure 2: Two primary antibodies with the same isotype can ...be converted? Isotype conversion is a unique characteristic of recombinant antibodies. Recombinant antibodies...

...mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with...Nature biotechnology (2016). PubMed PMID: 26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human... et al 2016) revealed some of the biophysical parameters that can influence the HDR/NHEJ decision. Our...or double stranded donor DNA with other design parameters. Example single stranded donor DNA and a presumptive...

... be directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open... cloning grade DNA beta project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular...plasmid without spending time and resources in preparative experiments.  We’re excited to begin offering...etc. and/or directly long term storage. Every preparation is quality controlled through next generation...

...cell types will separate into distinct populations on a FSC versus SSC plot (Figure 2).  A user can then...B cell marker) to separate the B cell and T cell into distinct populations (Figure 2). An immunologist...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ...the desired parameter. A droplet containing a cell that is positive for a desired parameter is collected...both cell surface and intracellular proteins in parallel, then stain the surface protein first, fix and...

...imaging of diverse tissues. Nature Protocols, 17(2), Article 2. https://doi.org/10.1038/s41596-021-00644-9 ...directly from the Zenodo dataset.     Figure 2: Screenshot showing reagent entries with associated... multiplexed: you can image over 65 different parameters using this method (see Movie S8 from Radtke et...

...target DNA strand separation is stabilized (2,3). Stable strand separation is maintained by 2 sets of interactions...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ...basal strand separation. This decreased strand separation combined with the weak separation afforded by... 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation is maintained...alanine substitutions throughout the groove in 32 separate Cas9 mutants. When tested for their ability to...M694A/Q695A/H698A). This new variant displays comparable on-target activity and similar or decreased off-target...

...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA... had a similar level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image...Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073/pnas.76.2.615  Zhou...rather a glass powder slurry that has a milk-like appearance. To make DNA stick to the glass particles in ....0007750  Vogelstein, B., & Gillespie, D.A. (1979). Preparative and analytical purification of DNA from agarose...

...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...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...

...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger... purified and labeled separately with different dyes. b) mDia1 and CP appearance at the filament tip from...the filament from (a-b), showing the appearance and disappearance of 649-mDia1 or 549-CP (upper, middle...

..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional single-stranded...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...names of AAVs contain two numerical designations, separated by a slash, that indicate the ITR and serotype...

...conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...a targeted neuronal population.    Figure 2 (modified from Schnutgen et al. Nat Biotech 2003):...cellular level in the mouse. 2003 PubMed PMID: 12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution... SM. 2008. A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...will be greatly reduced, allowing for clearer characterization of the location, morphology, and circuit mapping...

...the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The El-Samad...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for ...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...knockout techniques. Find the plasmids from Aranda-Díaz et al. Aranda-Díaz, et al. ACS Synth Biol. 2017. Pubmed...demonstrate independent control of gene expression, Aranda-Díaz et al transformed yeast with both a GAL4/estradiol...each other, these systems can be used together. Aranda-Diaz et al additionally show that it is possible...places reporter expression under the control of two separate synthetic transcription factors in series and ...

...AAV2 vectors in the mouse retina. Mol Ther . Feb;19(2):293-301. PMID: 21045809 Other citations include: ...sequencing of the final viral vector preparation. These viral vector preparations were produced with the pTR-UF11...Service , Addgene is producing ready-to-use viral preparations from the University of Florida for enhanced ...-UF11 plasmid ( Addgene #157970 ). Viral preparations are formulated in one of the following three buffers...injection. It was identified by John Chiorini and characterized by Shannon Boye’s lab. AAV44.9(E531D) The AAV44.9... of glaucoma and their reversed cell entry characteristics. Gene Ther . 2020 Apr;27(3-4):127-142. PMID...Shannon Boye and cite Boye, et al. 2016. Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant...

...FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) and synaptic...Sternson. 2008. “A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range ...PMID: 18614669. PubMed Central PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart....The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl...structure of 13 base pair palindromic repeats separated by an 8 bp asymmetric core. Recombination between...recombinase. The high concentration of FLEx vectors guarantees that most cells are transfected with all labeling... knockout of a target gene and expression of a separate gene. In these mouse lines, gene knockout can ...

...increase multimer formation Δ(ara-leu)7697 ΔlacX74 ΔphoA PvuII phoR araD139 ahpC galE galK rpsL F′[lac+ ...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression...recommended for toxic proteins BL21-A1 Tetracycline Arabinose-inducible expression of T7 RNAP; IPTG may still... expression F- ompT lon hsdSB (rB- mB-) gal dcm araB::T7RNAP-tetA General protein expression  BLR ...expression strain to make sure they are compatible. An arabinose-inducible plasmid will not express in an IPTG ...