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We narrowed to 1,106 results for: Ide

Showing: 361 - 380 of 1106 results

  1. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ...trying to decide which CRISPR tools are best for your experiment, check out our CRISPR guide or leave ...can be used to modify any desired genomic target provided that (1) the sequence is unique compared to the...Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9...For more details on the PAM site, check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel...series of positive selection screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to ...Kleinstiver et al. 2015). From these screens, they identified four novel SpCas9 variants with altered PAM binding...Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome Biol 19: . https...

  2. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...experimental work, but with this guide, we hope you feel more confident incorporating them into your research... We strongly recommend following the protocols provided by the depositing lab whenever available. Addgene...library sample, and any other information or scripts provided by the depositing lab for data analysis.  I'm ... If not, our protocol on Lentivirus Production provides a general approach for producing lentivirus in... the virus–the right amount of virus, that is. Ideally, each cell will be transduced by no more than one...from Addgene? Take a page from our book and put aside an aliquot for titering and optimizing before you... number of cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing...

  3. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...as a newbie reprogrammer, to decide which method to use. This post provides a brief overview of reprogramming... them with peptides that help mediate their transduction, such as the polyarginine peptide described in...variation between iPSC clones. Retroviruses are not considered safe for clinical applications.     Lentiviral...Like retroviruses, lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox... site of excision, this approach is still not considered safe for therapeutic applications. piggyBac (...insertional mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors ... includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm...

  4. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman et al...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...popular tools for genetic screens, enabling genome-wide interrogation of the genes required for cellular...processes. For a successful screen, it is necessary to identify the gRNAs that elicit the phenotype, and this ... the gRNAs responsible for a phenotype can be identified by next-generation sequencing of the genomic ...contributed by Leah Schwiesow A leading cause of death worldwide, cardiovascular disease is driven by atherogenic...ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents (what is commonly called...

  5. With Our New Viral Service, We're Taking CRISPR Further!

    Type
    Blog Post
    ...excited to provide ready-to-use CRISPR lentiviral preps to researchers across the globe. Why provide CRISPR...for genes involved in a wide variety of cellular processes. While these are widely requested and incredibly...example, we’ve teamed up with the Broad Institute to provide lentiviruses for delivery of SpCas9 and Cas9 nickase...BRAF and MAP3K4 gRNAs allow you to target these widely studied signalling pathways. Find Validated gRNAs...alleviate some of this labor for our users, we now provide the Brunello (targeting human genes) and Brie (...gRNAs to target around 20,000 individual genes. We provide enough lentivirus for you to both perform an optimization-scale...lentivirus stocks. Our research scientists have provided their standard lentivirus production protocol ...

  6. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...scatters sideways to the path that it was initially traveling is called side scatter (SSC) and provides information...their observations. However, while flow cytometry provides analysis at the cellular level, it cannot be used...degree of scatter is captured by detectors and provides useful information about the cell. Light that ... traveling is called forward scatter (FSC) and provides information about the relative size of the cell...reporter on the N- or C-terminus of the protein. One downside of fusion proteins is that the tag can alter the...granulocytes in a plot of forward scatter (FSC) versus side scatter (SSC). Users can gate around their desired... analysis, include a complete set of controls. Ideally, every flow cytometry experiment will have a negative...

  7. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ...appropriate for your specific research question. Considerations when choosing your Cas nuclease When choosing...experiments, there are three important factors to consider: site accessibility, specificity, and sensitivity...cleaves a site that is not a perfect match to the guide RNA (gRNA) used to target the genomic locus of interest...interest. As these off-target effects can be genome-wide and, in some cases, difficult to determine, the ... off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity...measured by calculating the on-target activity divided by the off-target activity. Figure 2: Specificity...engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al., 2018). The David Liu...

  8. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    ...Madison lab created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA...well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using...fluorescent reporter. Lastly, once a mutant is identified, the PB transposase can easily and seamlessly...fluorescent protein for assessing CRISPR/Cas9 activity Guide RNA structure and the site in the genome you are...Sander and Joung, 2014). Thus it is essential to identify the best sgRNA to ensure success in your CRISPR...-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired ...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of mCherry...

  9. Advice for Starting a Biotech Company from a Grad Student turned Entrepreneur

    Type
    Blog Post
    ...himself from the idea of starting a company around engineered bacteriophages. He decided to quit his "safety...Operations of Sample6, for advice on how to take an idea developed during graduate school and turn it into...into a biotech company. Transitioning an idea from academia to a startup Mike Koeris and fellow co-founders...co-founders Tim Lu and others first came up with the idea that would evolve into Sample6 as graduate students...using bacteriophages as bacterial therapeutics. This idea was incorporated into Novophage Therapeutics, Inc...that entrepreneurs must clearly communicate their idea at various levels of complexity and abstraction ...people with diverse backgrounds how amazing their idea is. While communication styles in academia and industry...

  10. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ...structure of the POI would work best. Ideally, structural information can guide this decision. In our experience... This is an important downside of the technology and should always be considered when interpreting results...that is strong evidence that the fusion behaves well. But this is not conclusive evidence that the fusion... any tendency of GFP to homodimerize should be avoided because it will double the size of the protein....or (ii) designed to form an inert, unstructured peptide and therefore consisted of glycines, serines and...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...al., 2011).  Although structural information may guide design, it is advisable to generate multiple constructs...

  11. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    ...size, the cytometer provides you with two measurements: forward scatter (FSC) and side scatter (SSC). To... being considered for the analysis. (C) A more liberal gate allows more cells to be considered for the... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage...scattered forward at a low angle (FSC) or to the side at a large angle (SSC). FSC and SSC depend on the...three gates will likely be: the FSC/SSC gate to identify cells, the single cell gate to exclude duplicate...running my flow analysis, I can however clearly identify my lymphocyte population (10.7% of all recorded...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...

  12. Click Biology: How to Click and Play to Explore Biology

    Type
    Blog Post
    ...and Golgi apparatus and outside the cell, most cysteines are oxidized in disulfide bonds. Reactions also...between a genetically encoded peptide (DogTag) and protein (DogCatcher); ideal for protein loop regions ...child (some of us still do), so we are used to the idea of general interfaces and the freedom to test out...2025). Figure 1: Modular assembly tools, alongside criteria for Click Biology. Created with BioRender.com...Click Chemistry reactions, one component bearing an azide reacts covalently with another component bearing...nucleic acid sequence (Addgene can help with this!). Besides requiring stable covalent reactions, other required...required parameters of the coupling system include: Wide fusion-tolerance — Click Biology tags should be ...

  13. Human Germline Editing Using CRISPR

    Type
    Blog Post
    ...clinical applications could be considered. It is important to consider the question of broader applicability...underwent repair not from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but...allele rather than the supplied single-stranded oligonucleotide (ssODN) template. Embryos also underwent editing...failure of the embryos to undergo editing using the provided ssODN template. In fact, this finding led the ...conducting research in embryos should our society decide that germline editing is permissible in cases of...disease prevention. However, it is important to consider that already-tested CRISPR alternatives are applicable...target. In addition, as on-target editing rates vary widely from gRNA to gRNA, it’s unclear where this study...

  14. Addgene's Tips for Plasmid Quality Control

    Type
    Blog Post
    ...week. Here we will provide an inside look at the steps we take to verify the identity and quality of the...plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through...the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids... ends to verify that the sequence assembly and identity of the insert are correct. In other cases, we ...glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then select forward...corresponding glycerol plate on dry ice to the sequencing provider. Each sequencing service will have different requirements...requirements for volume and concentration, and not all providers are equipped to perform Sanger sequencing from...

  15. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    ...to start considering my future path. Talking to Addgenies has helped me get a much better idea of what ...organization beyond my job description.” Our resident bagel provider, source of energy, and conversation master...friends in the Biochemistry major seem to have the idea that if you don’t get your PhD you’re set up for...undergraduate school and studying for the MCAT, she decided that she didn’t want to take it. “I realized if...to her. Addgenie Shreya Vedantam with our resident dinosaur! Read her Instagram feature here. ...scooped, she said. Reflecting on her values, she decided that this “is not [her] view of science. It should...joined an investment club and discovered the business side of science, she stuck with it. Lab Manager Shannon...

  16. Design Tips for Prime Editing

    Type
    Blog Post
    ...Editing Guide RNAs Download the CRISPR 101 eBook Resources on Addgene.org Read the CRISPR guide Find CRISPR...tools. These papers are chock full of great tips to guide your experimental design, especially the design ...nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The pegRNA consists of a CRISPR gRNA...successful, although sequences with G/C content outside this range can still be optimized (Anzalone et ...transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even more important...influence the success of your prime edit. If possible, consider following these suggestions: Edit the PAM along...DNA mismatch repair (MMR) is less efficient at identifying these “bubbles” of mismatched bases, enhancing...

  17. Tips for Technical Support Calls

    Type
    Blog Post
    ...get an answer, consider asking to avoid unrealistic expectations. And finally, provide feedback. If ... in customer service, I've daydreamed about the ideal customer service interaction - one that gives our...experience is based solely on my time at Addgene, I’m confident that these tips and tricks will apply to any biology-related...Your time is precious. Here are a few things to consider before you commit: Double check to make sure ... it’s not in the best interest of any company to hide information about their products from you, unless...papers that successfully used said plasmids. We provide these references as our way of saying “Hey look...can be a particularly good resource if you are considering using a reagent for “off-label” purposes. When...

  18. CRISPR Kinome Libraries Available: Pooled and Individual Plasmid Formats

    Type
    Blog Post
    ...individual gRNA from this single vector) and guide-only (lentiGuide expressing only an gRNA, for use with cells...new model or a new assay or both, and you have no idea what genes are involved. Or you don’t know how well...Or you don’t have enough cells to execute genome-wide perturbations. So, while some might argue that the...the CRISPR kinome library? Thus, we are glad to provide CRISPR libraries targeting the mouse and human ... a well-annotated collection of genes that can provide a useful starting point for developing a model ... libraries are available from Addgene as pools provided in two sets, each with 4 gRNAs per kinase, so ...opposed to the 70,000 or more gRNAs found in genome-wide libraries, and thus can be screened in pooled format...

  19. The Scientific Conference Poster Session: Tips for Success

    Type
    Blog Post
    ...with people inside and outside your field. Your colleagues, lab mates, and PI can provide feedback on ...poster – now it’s time to practice your delivery. Consider your audience: Who will be attending the session...audience so that they can digest what you’re saying, provide constructive criticism, and ask thought-provoking... for Cancer Research Annual Meeting, you might consider including more background on how your virus of... points will help you remember what to say and provide your audience with a clear picture of your work...on your technical presentation, but those outside of your field can point out places where you gloss over...you’re standing at the session. Present with confidence Now that you’ve printed your poster and practiced...

  20. PCR Applications for Copy Number Variation Assays

    Type
    Blog Post
    ...though it has been shown to identify trends correctly (Muhamad, 2011;  Providenti, 2006). Its biggest advantage..., look for the lowest-cost option that provides you confidence in the results you need.  Whichever ...Taqman’s increased sensitivity allows for a more confident determination of copy number, albeit within a ...to a standard curve, and most results can be confidently interpreted as integers. Since each sample is...approach is right for your experiment. You’ll have to consider:  Resources needed Most molecular biology labs...exact copy numbers or just general trends? How confident do you need to be in your determination to move...determine exact copy number and it may not be able to identify trends in cases where the copy number variation...
Showing: 361 - 380 of 1106 results