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Showing: 241 - 260 of 1105 results

  1. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...immunohistochemistry, and are summarized in the table below:   ID Cv (Capsid) FLAG Tag Terminus 79629 (N) and 79628... Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-...DHS-seq, FAIRE-seq, and CHIP-seq), genome-wide identification and quantification of enhancer activity have...RNA-guided targeting mechanism. However, the laborious construction of repair templates and guide RNA ...: Targets of RNA-binding proteins identified by editing Identifying targets of RNA-binding proteins (RBPs...region sequencing) that tests enhancer sequences outside of their endogenous chromatin environment and relies...screen the entire genome for genes that regulate a wide variety of phenotypes, including but not limited...

  2. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169...Cas9, been identified. In Rauch, et al. 2016 the Joseph Bondy-Denomy lab at UCSF identifes four proteins...our hot plasmids articles. These brief articles provide a synopsis of a plasmid or group of plasmids' functions...may be preferred for their intended uses, they provide a well-defined framework for the production of ...available at Addgene, and the Doyon lab has also provided a detailed protocol at Protocol Exchange. Plasmid...editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated with... created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9. They subsequently tested...

  3. Video Library

    Type
    Protocol
    ...questions. Protocols Video protocol guides for standard laboratory procedures. How-To Videos Screencapture walk-throughs...Protocol How-To Videos (Link opens in a new window) Video Link Description Related Page MTA Guide - Requests... Educational Resources Addgene Videos Addgene Videos Browse Addgene's collection...collection of video content. Find video protocols, tips on using Addgene's resources, and career advice. For...walk-throughs to help guide you through Addgene's online resources. Career Videos Get career insight and...to Deposit Your guide to depositing plasmids with Addgene Depositing Page Career Videos (Link opens in ...A collection of Addgene's video content, including how-to screen captures, lab procedure protocols, and...

  4. CrispyCrunch: High-throughput Design and Analysis of CRISPR+HDR Experiments

    Type
    Blog Post
    ...(See section below.) For guide design, we decided to adapt the popular guide design tool Crispor by Max...which the guide binds. However, if the PAM resides in a coding region, the mutation may cause side-effects...optimal mutation in CrispyCrunch: If the guide PAM resides outside a coding region in a UTR, CrispyCrunch... experiments designed outside CrispyCrunch, or you can input pre-existing guides for primer design. The...return guides and primers in batch, optimized for HDR. Compared to wealth of resources for guide design... summary per batch. Ranking guides for HDR in-depth In other tools, guides are ranked by specificity of...specificity of a guide to the target. The CFD score ranges between 0 and 100 for each guide, with 100 being...

  5. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    ...genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by sequencing (GUIDE-seq) ... Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing Provided 20% ...PMC3782611. 8. Tsai, Shengdar Q., et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by ...gel electrophoresis. Surveyor™ and T7E1 have been widely adopted due to their relative simplicity and low... problem with these assays is that they do not provide sequence-level data. They also have a limit of ...sequencing. Used in concert, these approaches can provide the researcher with a reasonable level of certainly...off-targets. This is a valuable step toward enhancing confidence and reproducibility of a study’s findings. Table...

  6. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...Views Guides CRISPR Guide Lentiviral Guide AAV Guide Optogenetics Guide γ-Retrovirus Guide Protocols... popular videos are protocol videos, where showing exactly how to execute a step can provide needed clarity...accessible, due to a wide range of logistical barriers. Addgene worked to identify, reduce, and remove ...allowing people to access a wide catalog of plasmids from all over the world. This idea, that available and accessible...Addgene distributes to per year, 2005-2023. We identified and offered a number of solutions that increased...over half of our orders coming from researchers outside the USA, having some materials available to order...consistently accessible, wasn’t an option. So we identified countries that would benefit from a third-party...

  7. A Look at Addgene's QC Process

    Type
    Blog Post
    ...plasmids we distribute to scientists worldwide. Depositing scientists provide different types of data for each...compared to the information provided by the depositor. If a depositor provides a sequence for the plasmid...sequences without a reference; and identifying mixed plasmid and nucleotide populations. All of these topics...if one was provided. If no reference is given, de novo assembly in Geneious can still provide additional... their presence with confidence. If this approach does not resolve the identified differences, the plasmids... both human and biological. Even the most high-fidelity polymerases can make mistakes and lead to the ...plasmids in our collection. This standardization provides security for requestors, ensuring that the plasmid...

  8. Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

    Type
    Blog Post
    ...DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the RNA guides employed...remains faithful to its guide DNA and cannot switch guides. While this fidelity would prevent other small...24 base DNA guides may be more user-friendly, since they can be ordered as oligonucleotides and should ...gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA guides must ... tested if NgAgo preferred certain guide sequences - using 5 guides/gene for 10 genes, they did not observe... the DNA guide impaired NgAgo activity, with three mismatches abolishing activity. One-guide faithful...important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also...

  9. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...similar to TIDE known as TIDER (Tracking of insertions, deletions, and recombination events). TIDER can screen...both trace files to the TIDE tool online along with your sgRNA sequence. The TIDE software will generate...off-target effects with Sanger sequencing, provided you have some idea of where in the genome you might expect...knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et al., NAR). This technique uses the... deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files of WT and edited...input population or clone.   How to perform a TIDE First, use PCR to amplify the target region of interest...graph representing all insertions and deletions identified within a given genomic window, along with the...

  10. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...with peptide/protein analyses using ELISA. Peptides 72:4–15 . https://doi.org/10.1016/j.peptides.2015.04.012...vary the acrylamide content of the gel. For small proteins, use a higher percentage of acrylamide to increase...usually composed of a nitrocellulose or polyvinylidene difluoride (PVDF) in a step called protein transfer...they provide qualitative measurements. If you require precise quantification of a protein, consider using...immunoprecipitation and pull-down assays.  Westerns are divided into two categories, native and denaturing. In ... protein mix by size Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first...agent, typically containing thiols, to cleave disulfide bonds. Load the samples, one sample per lane, ...

  11. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ...Having answer slides ready after your formal conclusion slide will also help with your confidence. It is best...suspense. Notice the simplicity of their slides and how the slides complement the spoken message. Learn from...speak to start creating slides or thinking about content. Create a Google.slides or Powerpoint file right...this might sound condescending, consider explaining the term on a slide but not reading the explanation...everything I allow 1 slide for every 1 minute of talk length, and then I cut a few more slides. Do not run over...always be a future boss in the room, so it is a good idea to start thinking about public speaking early and...talks for non-academic settings. Many of these guidelines will apply to any type of science presentation...

  12. High Complexity Golden Gate Assembly with NEB’s DAD

    Type
    Blog Post
    ...overhangs with the same three nucleotides in a row; (4) no more than two nucleotides in the same position; (...that high-fidelity reactions could be done using overhang sets that broke rules 3—5. Fidelity, it turns... design high-fidelity Golden Gate Assemblies and overhang sets: NEBridge Ligase Fidelity Viewer™; NEBridge...reactions. NEBridge Ligase Fidelity Viewer This tool allows a user to check fidelity of an overhang set by ...remarkably high 71% fidelity, while the 52-piece assembly of a lac operon dropped to 49% fidelity, with significant...be strictly followed in order to generate high-fidelity reactions. (Need a refresher? The five rules are... a single tube. Their assembly had a predicted fidelity of 71% (Pryor et al., 2020). And in 2022, using...

  13. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    ... requires that you provide two out of six forms of documentary evidence: Evidence of receipt of major...obtain permanent residence. The recently established case Matter of Dhanasar provides the three-prong ...field Evidence of original scientific or scholarly research contributions in the field Evidence of authorship... to convey general information only and not to provide legal advice or opinions. The contents of this ...advice on specific legal issues.  The permanent residence “green card” process is a necessary and often ...uncertainty associated with filing a permanent residence case with United States Citizenship and Immigration...national researchers who require visas, permanent residence or citizenship to continue their work in the US...

  14. Of Myc and Men

    Type
    Blog Post
    ...defined peptides. Munro and Pelham’s first published peptide tagging experiment involved a peptide from ...with unrelated peptides that already had high quality antibodies available - aka peptide tagging.  Key ...bound to the mid-protein peptide, while three bound to the C-terminal peptide. One of these C-terminal... of these new antibodies and identified Myc1-9E10 and its target peptide as a suitable pair for protein...from the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino...could be quite small. However, substance P has an amide group added to its C-terminus post-translationally...critical for antibody recognition. As such, the peptide could only be added to the C-terminus of proteins...

  15. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking. The...One 2013 Oct 7;8(10):e76117. Irreversible peptide-peptide ligation using SpyLigase Building off their...that promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against...scientists to use CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries... to browse CRISPR plasmids, watch informational videos, download protocols, and more! Looking for background... CRISPR technology? See our improved CRISPR-Cas Guide. Wang et al., Science. 2014. Jan 3;343(6166):80...

  16. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ...libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined ...10-6 mutations per nucleotide per generation vs. 10 x 10-10 mutations per nucleotide per generation of ...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...O., et al. "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window... changes to a DNA sequence can help scientists identify and/or facilitate the evolution of new phenotypes...method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...

  17. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    ...even single mismatches within a CRISPR guide can prevent the guide from binding to the genome or prevent...thetargeting sequences of guides across 8 CRISPR libraries. Samples are divided into those of African ancestry...approximately 20% more single nucleotide polymorphisms (SNPs) that map to guides in the Broad’s Genomic Perturbation...accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’ genomes... CRISPR screens in DepMap to see if they could identify the ancestry of the cell lines (European, East...germline genetic variations. They then wanted to identify cancer-associated genetic dependencies associated...relationship between the genetic dependencies they identified and their associated ancestries. While there ...

  18. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...adapted "self-cleaving" 2A peptides into their muliticistronic vectors. These peptides, first discovered in ... these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at...cleavage with some of these peptides, no consensus has been reached on which peptide works best. Likely the...from a single transcript. The two strategies most widely incorporated into plasmids for research purposes...the specific genes cloned into the vector.3  2A Peptides To overcome some of the disadvantages of the IRES...between the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the ...Proline.  The table below lists the four common 2A peptides employed by scientists. 2A cleavage is universal...

  19. Tips for Using BLAST to Verify Plasmids

    Type
    Blog Post
    ...menu. Typically, we use the default nucleotide database “Nucleotide collection (nr/nt)” as it contains ... the Basic Local Alignment Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing...reference sequences, such as full plasmid sequences provided by the laboratories that deposit their plasmids...programs available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp...BLAST (blastx). NCBI has a terrific getting-started guide for BLAST, which includes a simple explanation of...BLAST search pages. At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as...ORFs) and determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs...

  20. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...A,T or C – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations...mutation), blocking the guide RNA binding site works well. However, since some guide RNAs may tolerate a ...end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred...repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the intended sequence...efficiency.   But what’s better: PAM-blocking or guide RNA blocking mutations? This depends entirely on...however we have also demonstrated the efficiency of guide RNA blocking mutations. So if the locus does not...while taking advantage of the improved efficiency provided by blocking mutations. Essentially, you:  Step...
Showing: 241 - 260 of 1105 results