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We narrowed to 968 results for: LAT;

Showing: 161 - 180 of 968 results

  1. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...target in the HR template, you usually would need to make mutations of the HR template to avoid this donor... HR template an issue? A8: Yes. If you have intact protospacer+PAM sequence within the HR template, it... that if a CRISPR is used to engineer a locus, isolate single cell colonies, then genotype them, it is...homologous recombination (HR) Q7: Can you summarize HR template vector design guidelines? A7: A few notes below...you should consider using ssDNA oligo as donor template, it's usually more efficient than plasmid donor...µl of 10 µM oligo stock for each well on 24-well plate, co-deliver into the cells with the Cas9/sgRNAs ...with Cas9/sgRNAs vector, for one well in 24-well plate. A good example/reference is Ran et al. (2013). ...

  2. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    ...the model organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows...but currently requires additional experimental manipulation. In some cases, injection of Cre is required...mutation must be removed, which can be done by isolating animals with particular visible phenotypes. These...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits to only four...but require variable amounts of experimental manipulation and screening.  A co-selection strategy for ...be obtained in eight days. The only animals on a plate, other than the parental animal, are rescued progeny...

  3. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ...developing a general translation initiation sequence known as the Species-Independent Translational Sequence (SITS... fuse custom PUF proteins to a translational repressor to down-regulate a gene of interest. You can generate...: 1147. Cooke, Amy, et al. "Targeted translational regulation using the PUF protein family scaffold....extensive sequences used for cloning into the final translated products. Negative selection markers built into...Tikhonova, Elena B., et al. "A transcription blocker isolated from a designed repeat protein combinatorial library...of which you can clone your gene of interest. Translation of the standardized leader peptide prevents secondary...encoding your gene of interest from affecting its translation initiation and instead makes initiation heavily...

  4. Plasmids 101: Screens vs. Selections

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    Blog Post
    ...Forward and reverse screens involve manipulating or mutagenizing a population (typically cells or small organisms...screens, there is random or unknown manipulation of the population. This is contrary to reverse screens...disease as the input population. For these experiments, the goal is to identify manipulations which either enhance...in that only survivable manipulations are assessed, rather than all manipulation events. To put it another...looking at cells that can survive. Instead, all manipulations are assessed, regardless of whether they enhance... is assessed. The two most common methods of manipulation are genetic knockout mutations and treatment...consider these questions to save yourself a headache later on. Enhancer and suppressor screens These types ...

  5. Hot Plasmids: Spring 2025

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    Blog Post
    ...TurboCas, the Ali Shilatifard Lab was able to identify both new and known regulators at the FOS gene promoter...nearby proteins with biotin. The biotinylated proteins can then be isolated and identified with tools like..., the Shilatifard Lab created a very quick and efficient way to learn more about gene regulation. Find...Research Campus (Aggarwal et al., 2025).In this latest work, the team designed and screened a large library...making it ideal for video recordings of large populations of synapses and deep-brain fiber photometry.  ...mammalian expression and AAV plasmid sets launching later this year!  Find Addgene's own Lentiviral backbone...Glypicans By Ashley Waldron Glypicans are important modulators of cell signaling pathways during embryonic development...

  6. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    ...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...particular organism. Why is codon usage important? Translation and protein synthesis relies on transfer RNAs...efficiency is correlated with codon bias across all endogenous genes in E. coli and S. cerevisiae (Tuller et.... Image from Mariana Ruiz Villarreal. By the late 1980s, scientists even created a codon adaptation... 2000 the Ikemura lab created the Codon Usage Tabulated from GenBank database (CUTG), that provides an...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...an organism that contains expression limiting regulatory elements or use a non-canonical code (Gustafsson...

  7. New Tool for Lineage Tracing: The ClonTracer Library

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    Blog Post
    ...the resistant populations. In fact, 88% of the barcodes within the resistant populations were shared by...screening with lentiviral pooled shRNA libraries. Later, Bhang says, “When we developed this tool, there...library to determine whether or not cancer cell populations resistant to drug treatments arise during treatment...treatment or are preexisting in the parent population prior to the start of treatment. They hypothesized...hypothesized that, if they labeled the parent cell population using the library, propagated it, separated it into...cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates...that are present at less than 0.1% of the total population are easily be missed. The ClonTracer library ...

  8. Early Career Researcher Toolbox: Free Online Molecular Biology Tools

    Type
    Blog Post
    ...ratio calculators Lab math calculators These calculators can help you with common calculations performed...lab. Molarity calculator Promega's Biomath Calculators Primer resuspension calculator (sign-in required...sequences to sequences in a database ExPASy translation tool: Translates a DNA sequence using all six open reading... Cloning ligation reaction calculators These links can help you calculate how much insert and backbone...Annotates features, BLASTs, aligns, digests, and translates a sequence.   NEBCutter2: For finding restriction...information, such as expression patterns, post-translational modifications and polymorphisms Codon optimization...Addgene's Analyze Sequence tool.   DNA sequence manipulation tools  Sometimes DNA sequences need to be “cleaned...

  9. Plasmids 101: Common Lab E. coli Strains

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    Blog Post
    ... mk+) Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not...from two individual isolates, the K-12 strain and the B strain. K-12 was isolated from a patient in 1920...pathway that cleaves methylated cytosine DNA Allows for uptake of foreign (methylated) DNA mrr, Δ(mcrC-mrr...that cleaves methylated adenine or cytosine DNA Allows for uptake of foreign (methylated) DNA recA, ...plasmids that should not be Dam or Dcm methylated, allows for methylation sensitive restriction enzymes to ...renaming samples throughout history. It was likely isolated in 1918 but was first referred to as “B strain...routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that make these...

  10. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    ...strands of the template DNA first have to be separated. This occurs by heating the dsDNA template to a point...categories of multiplex PCR:  Single template PCR reaction - one template is amplified using several forward... reverse primer sets.  Multiple template PCR - multiple templates with different primer pairs that align...invented in the early 1980s by Kary B. Mullis who later shared a Nobel Prize in Chemistry for his work. ... in temperature. Taq was first identified in the late 1960s during research at hot springs in Yellowstone...polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique single stranded... primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...

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

    Type
    Blog Post
    ... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template. Although...laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing...from low efficiency when a standard dsDNA repair template is used. The original design for creating a conditional...targeting two intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand...ameliorate this problem. Briefly, you design your donor template to include a T7 promoter upstream of the left ...vitro transcription, and use the IVT product as a template for reverse transcription to create your ssDNA...

  12. RNA Extraction Without A Kit

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    Blog Post
    ...As with DNA isolation, scientists commonly rely on RNA isolation kits to make their life easier. Recently... out. In this article, we cover the basics of isolating RNA without a kit. Tips for working with RNA (... today There are many alternative methods for isolating DNA without a kit. However, that isn’t the case...method. A major hurdle to developing protocols to isolate RNA was that RNases are commonly found in cells...upon cell lysis, RNA is degraded. To effectively isolate intact RNA, a fast, strong protein denaturant would...chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues showed that a strong...., 1979). They developed a protocol meant for isolating RNA from rat spleens in which they homogenized...

  13. Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)

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    Blog Post
    ...detect proteins within a 96- or 384-well, flat-bottomed plate. The basic principle of an ELISA is that ...technique works by first coating the wells of a flat bottomed plate with a capture antibody that is specific ...experimental sample are bound to the wells of a plate instead of an antibody. Coating the wells with a...characterize antibodies against the antigen bound to the plate. Figure 3: Overview of using Direct ELISA...protein antigen is coated onto the wells of the plate. However, instead of detecting your protein using...absorbance of each well, which is measured using a microplate reader, is directly proportional to the amount... of enzyme present in the well and is used to calculate the amount of your protein of interest that is...

  14. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    ... the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long biotinylation targeting...Jul 2, 2020. Promoters may be the star of gene regulation, but enhancers and chromatin looping play important...important supporting roles. Enhancers are cis regulatory DNA sequences that, when bound by transcription...thousands of base pairs away from the gene they regulate, but are brought in proximity by the looping of...CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The original CAPTURE method...2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original CAPTURE method The original...the dCas9 to the loci of interest and dCas9 is biotinylated by BirA. From here, the CAPTURE method is similar...

  15. Plasmids 101: Protein Expression

    Type
    Blog Post
    ...formation of disulfide bridges, glycosylation and acetylation to create functional, stable proteins. Protein...used to manipulate DNA), or in medicine (e.g. insulin). Proteins, unlike DNA which can be relatively easily...cells with your DNA template of choice and allowing these cells to transcribe, translate and modify your ...transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into polypeptide chains that ...proteins are then modified via an array of post-translational modifications including protein folding, formation...proteins from mRNA and the addition of post-translational modifications Learn more molecular biology ...mammalian proteins that require multiple post-translation modifications for proper protein function. Most...

  16. A Primer on Optogenetics: Introduction and Opsin Delivery

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    Blog Post
    ...antibody manipulation), then expression is likely sufficient for successful optical stimulation.  It is...Gore BB, et al. Manipulating gene expression in projection-specific neuronal populations using combinatorial...technique is remarkably straightforward to learn and relatively cheap (compared to many other technologies and...electrophysiology to prove that your optogenetic manipulations are generating action potentials (or lack there...assess the effectiveness of your optogenetic manipulations (though a reviewer will probably ask for electrophysiology...circuit of interest in promoting reward is a self-stimulation experiment wherein mouse initiated optogenetic...in a reward sensation and subsequent repeated stimulation. A spatial memory task, on the other hand, would...

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

    Type
    Blog Post
    ...possible to build gene regulatory RSMs (GRSMs, the subset of RSMs that regulate gene expression) that ...massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and...individual bacterial cells to form colonies on a plate and then we performed colony PCR on the register...sequencing.  A less straightforward, but more population-wide (as opposed to single-cell) approach is ...a particular application makes it difficult to isolate single registers or if it requires reading so many...necessarily have to be based on transcriptional regulation, as long as they can control recombinase activity...post-transcriptional recombinase activation via RNA toehold riboregulation could be a promising strategy for linking RSM...

  18. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...example Tuller et al. found that translation efficiency is well correlated with codon bias in both E. coli...therefore may need fewer resources for the entire translation process. For example, during high growth rate...rate conditions, E. coli preferentially upregulates production of tRNAs that recognize codons found in highly...tRNAs, different regions of the protein may be translated at different rates. The ribosome will move quickly...the ribosome stalls, this may give the swiftly translated regions a chance to fold properly. For example...with specific secondary structures in 10 closely related yeast strains. Adaptation to changing conditions...codon preferences online). Even if the gene is translated, it may not function properly. This is the result...

  19. Deciphering the Mysteries of Behavior with Viral Vectors

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    Blog Post
    ...an shRNA to downregulate expression of miR124a, a regulatory RNA that negatively regulates BDNF levels...Christian Lüscher. "Refining deep brain stimulation to emulate optogenetic treatment of synaptic pathology...their involvement is activating, inhibiting, or modulating. Viral vectors, in contrast, are considerably...considerably more versatile and specific in how they manipulate neuronal function, with far fewer side effects compared...animal has been exposed to morphine or other stimulating drugs, it develops an enhanced propensity for...connection between neuroinflammation and anxiety by modulating the production of the pro-inflammatory cytokine..., or passive floating on the water surface. The latter reflects a state of behavioral despair, and the...

  20. Antibodies 101: Flow Cytometry Controls

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    Blog Post
    ...differentiate the Ly6C negative population from the Ly6C positive population. Figure 2: An FMO gating...clear positive and negative population. If you’re unsure what your population will look like for a particular...Markers that define more specific cell populations, often used in the later gates, are more likely to require...or isotype controls, which we’ll describe a bit later. Single Colors Single color controls contain, as...artificial positive (bound) and negative (unbound) population. The benefit of beads is that they do not require...allows you to more easily and accurately gate populations that may otherwise be difficult to separate out...commonly used markers that define broader cell populations (e.g., CD4, CD8b, CD11b, CD19), are less likely...
Showing: 161 - 180 of 968 results