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We narrowed to 1,000 results for: Eras

Showing: 341 - 360 of 1000 results

  1. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ...Newman-Keuls method – More powerful. Moderate false negatives, moderate false positives. Duncan’s multiple...is being experimentally manipulated. Below are several core factors of statistics relevant for many downstream...Parametric tests  Statistical tests fall into two general categories: parametric and non-parametric. In this...variable. A two-way ANOVA could test the effect of temperature and humidity on an outcome such as total crop... one-way ANOVA can test either the effect of temperature or humidity on crop production.   MANOVA The ...example, a one-way MANOVA could test the effect of temperature on total crop production and the production window...A two-way MANOVA could measure the effect of temperature and humidity on total crop production and the...

  2. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ...translation by generating a frameshift mutation. The majority of cellular repair events will generate these types... a less frequent DNA repair mechanism in general. On average, it accounts for less than 10% of DNA repair...family member which generates sticky ends upon cutting. Cas12a may promote HDR by generating ssDNA overhangs...Taking the road less traveled and generating a knock-in cell line instead of a knock-out? We’ve got you...editing Targeted genome editing events fall into two general classes: knock-in and knock-out. The goal of a ...exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful HDR ...viability of your cells. Enrich S-phase cells HR operates predominantly in S and G2 phase of the cell cycle...

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

    Type
    Blog Post
    ... the notation works? The + operator denotes intersection and the – operator denotes set difference. That... ChiRP-seq RNA Hi-C DNA (genome-to-genome interactions) DNase-seq DNA (regions that are accessible... the foundation of modern drug discovery and therapeutics. We’ll have more to say about clinical applications...Poisson statistics, each sub-population has, on average, one distinct enhancer disrupted. As our ESCs die...differentiate over time, we periodically use next-generation sequencing to measure the relative proportion...apply this knowledge to create targeted disease therapies with minimal side-effects on healthy cells and...Hypersensitive Sites and Intergenic CRISPRs Yours truly generated the DHS data set by starting with all of the DHS...

  4. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...A7: A few notes below are considerations for designing HR donor. Generally if off-target (non-specific... and surveyor. Q15: I used DNA polymerase Takara Ex Taq™ DNA Polymerase for my genomic PCR, but couldn't...In our hands, Herculase II Fusion polymerase or Kapa Hifi Polymerase work very well. Some groups have ...concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately...additional PAM sequences should not be sufficient to generate off-target DSBs; there needs to be extensive homology...most efficient and easiest way is to co-transfect several plasmid together, with each plasmid having a targeting...purified oligo is fine based on new data). For general transfection, you could use 1 µl of 10 µM oligo...

  5. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ... are typically DNA preps, but we do distribute several of our most popular CRISPR pooled libraries and... A) Amplification, validation, and lentiviral generation of a pooled library. An aliquot of the pooled...experiments. Pooled library lentiviral preps are generally aliquoted to reduce the number of freeze-thaw ...libraries we amplify in house, but these are not general amplification protocols. Rather, each protocol ...amplified, you'll need to verify your library by next-generation sequencing (NGS). The representation of your ...our protocol on Lentivirus Production provides a general approach for producing lentivirus in Lenti-X 293T...you can transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells...

  6. Career Insights: Technical Support Specialist

    Type
    Blog Post
    ...including tips, advantages, and disadvantages of this overall rewarding role. Learn about Other Addgenies and...role in technical support? It takes some time (several years) to build up comprehensive lab experience...You will also help troubleshoot products, give general experimental guidance, and resolve complaints about...willingness to help others with their problems, a general positive attitude, and an open-minded personality...any incoming requests or problems - there is considerable diversity in the types of questions customers...will do well in a technical support role. What general characteristics do jobs in technical support have...covered by the lab based team as part of their general job duties. Bigger companies have dedicated technical...

  7. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ... 101 blog, we reviewed the salient features of several popular strains of E. coli for DNA propagation....DE3) F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) General protein expression BL21 (DE3) pLysS* Chloramphenicol...ompT lon hsdSB (rB- mB-) gal dcm rne131 (DE3) General expression; not recommended for toxic proteins ...lon hsdSB (rB- mB-) gal dcm araB::T7RNAP-tetA General protein expression  BLR (DE3) Tetracycline RecA-deficient...miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression  m15 pREP4*, ** Kanamycin ...induction system. The DE3 lysogen expresses T7 RNA polymerase (RNAP) from the bacterial genome under control...overexpression? The strains described above should generate sufficient expression levels for most purposes...

  8. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    ... for monitoring how both the brain and visceral organs interact during various behaviors. Either tissue...very cool tool to advance our knowledge of brain-visceral organ signaling. The problem  Previously, there...emit light for optogenetic purposes, measure temperature, study electrophysiology, and deliver small molecules...molecules.   Fun fact: A 1 cm block of polymer can generate many meters of fibers!     Fig. 1 - ...for gut implantation. The fibers they ended up generating were 10-15 times more flexible than the brain... while brain tissue is relatively static). To operate both types of implanted fibers simultaneously, ...multiple channels and can also record tissue temperature proximal to the implanted fibers. As important...

  9. Plasmid Cloning by PCR

    Type
    Blog Post
    ...use a high fidelity polymerase to minimize mutations. The fidelity of the polymerase becomes more important...You should select an annealing temperature based on the melting temperature (Tm) of the portion of the primer... million bp depending on the polymerase used. No matter which polymerase you use, it is important that...restriction site to improve cutting efficiency. You can generally add any 6 bases, but you should ensure that the...with 18bp of homology to the ORF). We now need to generate the reverse-complement of this sequence so that...that we can successfully amplify the ORF. You can generate the reverse-complement using existing software...you can use a pretty wide range of annealing temperatures, but you may need to increase your primer length...

  10. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ...localization, interaction, and expression. However, one caveat of FP-protein fusions (FP-chimeras) is that ...that they undergo normal protein turnover. FP-chimeras are continuously synthesized and degraded within the...488nm blue light. The T203H mutation in PA-GFP generates a chromophore population that is more neutral,...developed red fluorescent PA-FPs by performing several rounds of random mutagenesis screening for enhanced...formation of mEosFP’s chromophore requires a temperature below 30°C and this is not ideal for experiments...tissue than the UV light used by other PA-FPs. Several other irreversible PA-FP spectral variants have...choosing a PA-FP for your experiment, there are several parameters to consider, such as brightness, photostability...

  11. FPbase: A new community-editable fluorescent protein database

    Type
    Blog Post
    ... a DOI referencing primary literature, and every effort is made to moderate and cross-check all data with...affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many... Addgene blog – outlining various important considerations when choosing a FP, but much of the primary... making such a decision remains spread across literature in publications that introduce these tools. ..., and I collaborated with him on a web-based interactive visualization of FP attributes (which turned ...that allows users to see when a given page has unmoderated changes and to view previous versions.  Currently... with published literature as soon as possible after submission. Using FPbase The most obvious use case...

  12. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...your incubation temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a...background noise. Temperature and time You may need to optimize your antibody incubation temperature and time.... variants Check literature Posttranslational modifications Check literature Highly charged...said, normalizing to total protein expression is preferable, and more accurate, than normalizing to ubiquitously...recombinant multiclonal antibodies to increase overall signal from the primary antibody.  Pro tip! If ...common conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies... including trying longer incubations at both temperatures if needed. Washes Check your antibody manufacturer...

  13. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ...we’ll go through some of the most important considerations to make when planning and performing an IF ...IF experiment. What is immunofluorescence? Generally speaking, IF is an application in which antibodies...iBiology). IHC and ICC are essentially just more general terms that can also refer to assays using non-fluorescent... visualize their target. These antibodies are generated against a specific antigen and then conjugated... more common is that secondary antibodies are generally polyclonal, which means that multiple secondary...amplification. Another reason is cost effectiveness. Generating and validating primary antibodies is a time consuming...antibodies that exist and you can begin to see why generating fluorophore-conjugated versions for each primary...

  14. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...coming of age. Molecular Therapy : the Journal of the American Society of Gene Therapy, 17(8), 1316–1332. https...can be an advantage for researchers looking to generate long-lasting changes in cells. However, for some...plasmids. For simplicity, we’ll use the second-generation system as an example.  The lentiviral transfer...integration, two types of circular episomes can be generated through either homologous recombination or non-homologous...integrase-deficient lentiviral vectors? There are several reasons why you might choose an IDLV over a lentiviral...clinical settings, as evidenced by retroviral gene therapy trials for X-linked severe combined immunodeficiency... 2000s. These trials unfortunately resulted in several patients developing T-cell acute lymphoblastic ...

  15. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...biotinylation, the discovery of the biotin-avidin interaction and the uses of biotinylation in molecular biology... and energy transduction. Biotinylation is the general process by which biotin is added to a protein or...protein (BCCP). Discovery of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg ... of the strongest noncovalent protein-ligand interactions found in nature. Once the bond is formed it ...it is unaffected by changes in pH, temperature or organic solvents. Uses of biotin in molecular biology...scientists began to exploit biotin and its strong interaction with avidin for molecular biology and biotechnology...that are coated with avidin. The biotin-avidin interaction has also been adapted for use in immunohistochemistry...

  16. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...many distinct CRISPR therapies to match these different mutations. For each therapy, effectiveness and ...’s size makes it difficult to deliver via gene therapy, so researchers have set their sights on another...work with dystrophin exon skipping? For such a therapy to be successful, multiple requirements must be...minimal risk of off-target editing. In order for the therapy to persist over time, stem cell editing would be...in DMD, and it’s estimated that exon skipping therapies would be applicable to 80% of DMD patients. ...characterizes additional, unique facets of CRISPR DMD therapy. Long et al. show that AAV-CRISPR does not cause...represent a great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler...

  17. Q&A with Karen Katz and Carl Paratore, the Newest Members of the Addgene Board of Directors

    Type
    Blog Post
    ...Brookline Interactive. Learn more about Carl Paratore, MBA, CPA Mr. Paratore serves as the General Auditor...Suffolk University Law School and Paratore is the General Auditor at Tufts Health Plan. The addition of Katz...perspective, what do you think are important considerations for the future of open science? What about ...University Law School. Suffolk University graduates on average 20 PhD’s a year and we have a very dominant presence...presence in the biotech sector in both patent law and general life science regulatory law. My role at Suffolk...work at Tufts Health Plan and my role there is general auditor. I oversee a department of 20 individuals...the efficiency and effectiveness of the Plan's operations. We’re looking for those “what could go wrong...

  18. Building and Validating Recombinant Antibodies: A Case Study

    Type
    Blog Post
    ... used to generate cDNA, which  was used as a template for PCR amplification with a degenerate primer set...limitation of hybridoma generated mAbs is that if the frozen cell line becomes non-recoverable, the stock of the... distributed by Addgene.  The plasmids used to generate these antibodies were based off of a large collection... they were also able to isolate RNA from non-recoverable hybridoma stocks, without having to grow up the... the cells. This RNA could then be used to generate functional recombinant monoclonal antibody (R-mAb)...manner. Cells were labeled with both a hybridoma generated mAb and the R-mAb, with the R-mAb being verified...encoding sequence within the plasmid backbone to generate R-mAbs of different subclasses, including subclasses...

  19. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    ...induced binding. The lab used this general design scheme to generate two more OptoNBs, one against mCherry...loop 1 always generated OptoNBs with dark-induced binding, but insertion into loop 6 generated both blue ...purification. After binding YFP to the column, you wash several times to remove non-specific proteins, and then...on the key components of OptoNBs, how they are generated, and their use for protein purification and controlling...chain antibody domain.  How do these components interact? When fused together to form an OptoNB, the AsLOV2...nanobody’s ability to bind its target protein.  Generating OptoNBs The opening and closing of the AsLOV2...over-activated in cancer due to its involved in cell proliferation and differentiation.  Since Ras is a membrane...

  20. Addgene's eBook Collection

    Type
    Guide
    ...and management, resources for identifying your transferable skills, and tips on the job application process...
Showing: 341 - 360 of 1000 results