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We narrowed to 955 results for: tes

Showing: 101 - 120 of 955 results

  2. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    ...have been used to visualize “thorny excrescence” (TE) spines in the Hippocampal CA3 pyramidal neurons ... The central nervous system (CNS) orchestrates complex processes enabling organisms to control their ... affects the labeling of small structures like neurites. Like epitope tags, low FP expression may be insufficient...structures through their multiple, high-affinity binding sites for primary antibodies which can be used to amplify...difficult to label. It has also been shown that dendrites in the stratum oriens and the stratum lacunosum...their multiple, highly specific antibody binding sites and primary antibody-based detection, smFPs create...

  3. Trimmer Lab NeuroMab Collection

    Type
    Collection
    ...mouse)/R. norvegicus (rat) rat IgG2a Addgene distributes ready-to-use recombinant antibodies made from...

  4. Ligation Independent Cloning

    Type
    Protocol
    ...digestion and PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in ...room temperature for 30 minutes. Inactivate T4 Pol by heating to 75° for 20 minutes. Volume (μl) Reagent ... for 5 minutes at room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at room...Protocols Overview Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step ...with a single enzyme, and remove the restriction sites from the final product (no "cloning scars"). A “...reaction may be stopped by heating to 75° for 20 minutes. Step 6: Anneal and Transform Mix your treated ...

  5. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    .... https://doi.org/10.1038/s41592-019-0614-5 Sztal TE, Stainier DYR (2020) Transcriptional adaptation: ...inverted repeats separated by a loop region that facilitates the formation of a hairpin RNA structure, resulting... gene of interest. Tools for RNAi Arguably the greatest advantage of RNAi is the straightforward nature...active development, the first step necessarily dictates the direction and scope of the subsequent two....Artificial micro RNA and short hairpin RNA The greatest limitations of the long hpRNA approach are thought...experiments typically involved antibiotic selection to test for the presence of the RNAi transgene, followed...Savitski M, Grandi P, Jakob P, Michon A-M, Sun H, Tessmer K, Bürckstümmer T, Bantscheff M, Steinmetz LM, ...

  7. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites (attL-left...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain modified...takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction...enzyme mix and generates the entry clone containing the DNA of interest flanked by attL sites. As a byproduct...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ... place between the attL sites of the generated entry clone and the attR sites of the destination vector...attB, P, L and R sites that recombine very specifically and directionally: attB1 sites react only with ...

  8. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...and astrocytes Mice No (LY6C1)   Hanlon et al., 2019 AAV-BI-28 CNS: neurons, astrocytes, oligodendrocytes.... It can also effectively transduce astrocytes or oligodendrocytes when paired with appropriate cell type-specific...28 has been tested in C57BL/6J mice and effectively transduces NeuN+ neurons and astrocytes (Huang et al...et al., 2023). Among the capsids tested, AAV9-X1.1 showed the greatest fold change in RNA and DNA levels... others have done, you’ll need to experimentally test your chosen capsid before using it in an application...elements [promoters and enhancers, miRNA target sites, and recombination target sequences, denoted by ...cisterna magna or lumbar spine. Image and caption courtesy of Challis et al., 2022. Where do they come...

  9. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    ... Cre but lox511 sites can only recombine with other lox511 sites, not with loxP sites. Alternatively, ...at target sites to induce site specific recombination events: Cre recombinase binds loxP sites, while FLP... FLP binds FRT sites. When a DNA sequence is flanked by target sites (floxed) in opposing orientations..., you could use a pair of loxP sites and a pair of FRT sites and include both Cre and FLP recombinases...proceed either by first utilizing the loxP sites or the lox551 sites. Regardless, the first recombination step...fragment using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment (Figure..., and that the orientation of the flanking loxP sites directs the Cre recombinase to invert, translocate...

  10. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    ...recombination one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure...orientation and one of the unused (loxp) sites will be flanked by two lox sites now in the same orientation. Next...recombination sites, they hypothesized that spontaneous recombination between these sites may be the cause...leaky expression. To test their hypothesis, they randomly scrambled one pair of lox sites and either used ...injection site. Test the promoter It is also essential that the promoter be thoroughly tested in your cells...fibrillary acidic protein (GFAP) for astrocytes it is important to test it in the brain region of interest...transgene is floxed by two pairs of recombination sites and the open reading frame is inverted in relation...

  11. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    ...do some testing or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell... the next gates fully depend on your individual experiment. Practical example: lymphocytes in a mouse...first three gates out of the way, it’s time to move on to my experimentally-specific gates. Two-dimensional...free to use multiple individual gates. Note that in this case, square gates were used.  Congratulations...distinguish single cells from duplicates. Lastly, you learned which gates should always be included in ...samples, it is necessary to set up a sequence of gates to be able to select and precisely measure your ...in cell size, marker expression, and viability. Gates are the parameters the machine uses to differentiate...

  12. Plasmids 101: Cre-lox

    Type
    Blog Post
    ...location and orientation of the loxP sites: Inversion: If the loxP sites are on the same DNA strand and are...region of DNA between the loxP sites is reversed.   Deletion: If the sites face in the same direction, ...If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites. How can...limitation of using loxP sites is the inability to tightly control which loxP sites recombine if more than...GtATACAT). These variant lox sites undergo recombination with other sites of the same type, but are not...inter-molecular recombination at the loxP recognition sites. A key advantage of the system is that Cre acts ...experiments. LoxP (locus of X(cross)-over in P1) sites are 34-base-pair long recognition sequences consisting...

  13. Hot Plasmids: Fall 2025

    Type
    Blog Post
    ...killswitch for biomolecular condensates By Emily P. Bentley Biomolecular condensates are membraneless liquid...approach eliminates the need for expensive reagents and equipment like incubators, micropipettes, and plate...Cell-surface protein labeling tools for neurons and astrocytes Nerve-to-cancer cell mitochondrial transfer tracker...FRCaMPi Micropeptide killswitch for biomolecular condensates AAV-CRISPRa-engineered fat cells fight cancer...classrooms Cell-surface proteomics for neurons and astrocytes By Mike Lacy Cell-cell interactions underlie ...important in the brain. To better understand how astrocytes and neurons interact, the labs of Baljit Khakh...extracellularly-anchored horseradish peroxidase (HRP) in striatal astrocytes and neurons to label surface proteins with a ...

  14. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    ...between the sites (Fig. 1B below). Alternatively, if an attB-attP pair is anti-aligned (sites facing opposite... their recognition sites, or by excising one or multiple of their recognition sites from the register..... Figure 1D below demonstrates an example of a register that enters distinct states when treated with ...recognition sites, terminators, promoters, and genes) were built with flanking primer binding sites (designed...registers. The primer binding sites can also be used to design primers to read states by qPCR or Sanger sequencing...that recognizes its own attB and attP recognition sites on the register and catalyzes their recombination...state of the RSM. If an attB-attP pair is aligned (sites facing the same direction) on a register, then the...

  15. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ... with three coordinates: chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of...notation works? The + operator denotes intersection and the – operator denotes set difference. That’s the ... critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen...all of the DHS sites from 125 different human cell types from the ENCODE Project. DHS sites are the most...as a kind of “GPS for the genome” to compute coordinates for genetic elements like protein, RNA, and DNA...DNA (Table 1). As with GPS in the real world, coordinates alone aren’t very useful. We’ll need something...come in. They help us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging...

  16. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    ...recognizes FRT sites and initiates site directed gene recombination between those sites. FRT sites and loxP ... of target sites that differ in their sequence. Recombination between the two target sites with identical...and the interior target sites. In a second step, the two compatible target sites are cleaved out, leaving...loxP sites differ at the nucleotide level but share an overall structure of 13 base pair palindromic repeats...bp asymmetric core. Recombination between these sites can lead to excision, inversion, and translocation...translocation of DNA in a similar fashion as between loxP sites (For details see Cre-lox blog post). Optimizing ...systems in your experiments. Mutated FRT and loxP sites in Flex Vectors – inversion only once All recombination...

  17. Getting Started with Behavioral Neuroscience: Standardizing Experiments and Using Viral Vectors

    Type
    Blog Post
    ...considerations for behavioral testing As a general consideration for behavioral testing in a laboratory, a high... some time to adapt to a testing apparatus. General stress levels of the test animal may also be reduced...AAV5 targets both neurons and astrocytes, and AAV4 targets mostly astrocytes. Lentiviral vectors are pseudotyped...weeks before the behavior test. This time window between injection and testing will vary depending on whether...influence on the outcome and cause variability in the testing group. For example, if an experimenter picks up... will likely move around faster in the following test and skew results measuring locomotion. Mice may ...reduced by daily handling for a few minutes by the experimenter several days preceding the experiment. Handling...

  18. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...bits (1.375 bytes) of information. This leads to 2048 (211) possible combinations of states. Each of the...protein binding sites and 24 copies of the MS2 bacteriophage coat protein binding sites to tether many ...reporter proteins that can be used to test diverse secretory routes in various conditions. The plasmids ...enzymes, such that the ligation regenerates the original restriction sites for further iterative cloning...2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA cleavage upon co-expression and spontaneous...which act irreversibly on their cognate attB/attP sites with no cross-talk. These pairs are closely related... in various combinations, while the cognate att sites are supplied as individual reporter constructs or...

  19. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand breaks...about 3 months. Image courtesy of Wenning Qin. Single-stranded DNA repair templates Short single-stranded...2 loxP sites to intronic regions 0.5 - 0.8 kb apart. In the example below, we’ll add loxP sites for conditional...disrupting splice donor/acceptor sites, Miura et al. (2018) recommend targeting sites at least 100 bp away from...place the loxP sites precisely at the cut site for each guide. This design also separates the gRNA target...single-stranded DNA templates have been shown to improve the efficiency of homology directed repair, with researchers...services or kits, including the IDT Megamer service tested by Quadros et al. When delivering the ssDNA template...

  20. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    ...cutting at mismatched guide sites, but not necessarily binding to these sites. Thus, these enzyme swaps ... ChIP-Seq, where only DNA sites bound by Cas are sequenced, has been tested with some success. The major... sequencing Sites with high sequence similarity to the gRNA can be sequenced. These sites can be selectively... this often means DNA cleavage at unanticipated sites. While off-target effects are nearly impossible ...or is engineered not to cleave DNA and instead activates/represses gene expression by various mechanisms...words, they can sometimes localize to unintended sites with sequence similarity to the on-target site. ...When a CRISPR-Cas system localizes to unintended sites and performs its programmed function there, this...
Showing: 101 - 120 of 955 results