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Showing: 261 - 280 of 895 results

  1. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...post has provided some insight on these differences in viral production and will help you decide which is.... In this post, we will compare two of the most widely used systems, mammalian and insect cells. Whether...cells are also easy to cultivate, fast-growing, and widely available. The viruses Lentiviruses, AAV, and adenoviruses...lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning..., they are easy to cultivate, fast-growing, and widely available. Unlike mammalian cells, they do not ... (about three to four days), this second round provides the added benefit of producing a stock P1 virus... your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in cell lysis...

  2. Pooled Library Amplifications

    Type
    Blog Post
    Published
    May 30, 2024, 1:30 p.m.
    ...important factor to consider is your desired library coverage, since this will help decide what transformation...and how will you ensure this?). You should also consider if there are any features of the library backbone...difficult to transform.    Figure 1: Factors to consider when creating a pooled library amplification protocol...amplification protocol is the Chong Zhang E. coli Genome-wide Inhibition Library. DNA and cell amounts After ... work! Selecting your media Lastly, you need to decide between growing your library in liquid media or...similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can...DOCX, 15 KB) CRISPR Activation Human Genome-wide CRISPRa-v2 Libraries 104,540–209,080 20 bp ...

  3. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    Published
    Aug. 3, 2023, 1:15 p.m.
    ... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...which spectral flow cytometry requires special considerations. Conventional flow cytometry During conventional...flow cytometry, light emitted by a fluorophore is guided to a single detector using a series of mirrors ...Instead of using a single (primary) detector to identify a fluorophore, spectral flow cytometers use all...thought of as a series of histograms flipped onto its side and compressed into a heat map. BV421 peaks in V1...cytometry for a given experiment, it is important to consider how many parameters you want to use in a given...and spectral flow cytometry, it is important to consider the expression levels of the markers you are including...

  4. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    Published
    Aug. 10, 2021, 1:15 p.m.
    ... or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR ...withstand changes in temperature. Taq was first identified in the late 1960s during research at hot springs...addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique...polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents and a series of heating...temperature is then raised slightly to Taq polymerase’s ideal temperature (70-75oC). At this temperature Taq polymerase...procedure check out our PCR protocol page, protocol video and reference on how to design PCR primers.  Types...PCR is used commonly in disease or pathogen identification. Scientists can simultaneously detect several...

  5. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    Published
    May 18, 2021, 1:15 p.m.
    ...lab provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...cell’s surface. The Bloom lab used the library to identify RBD mutations which do not interfere with ACE2....1074/jbc.RA120.01289     Novel protein cage provides simple enzyme protection in yeast bioproduction...enzymes in a reaction pathway, the MPyV system provides a simple, efficient method for protecting a single...- Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by anhydrous...anhydrous tetracycline. Three new anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein...

  6. Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

    Type
    Blog Post
    Published
    Dec. 10, 2020, 1 p.m.
    ...homologous recombination (HR). Adapted from an image provided by Manuel Gonçalves; made with BioRender. ...vectors As compared to other viral vectors, AdVs can provide a large transgene packaging capacity. For instance...chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in...in corrective genes. For this you would need to provide a DNA template to repair the double strand break...Nature Methods the same year that delivering RNA-guided nucleases or TALENs together with AdV donor DNA...demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available at ... Dystrophy, Alpha1-antitrypsin deficiency (most widely used so far) or for cancer gene therapy such as...

  7. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    Published
    Aug. 21, 2018, 12:31 p.m.
    ...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to easily ...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while...

  8. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    Published
    Oct. 10, 2023, 1 p.m.
    .... Amp is a semisynthetic derivative of the more widely known antibiotic, penicillin, and is a beta-lactam...buys you a little more time, since bacteria won’t divide immediately after it has been heat-shocked or electrically... it by producing a lot of beta-lactamase. Pros: Widely available, cost-effective, timesaving for transformations...Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits protein synthesis. The kan resistance...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...recovery period than amp or carb (60 minutes). Pros: Widely available, cost-effective, confers mammalian resistance... with all bacteria used in research, it isn’t a widely popular selection agent. Pros: Stable (spectinomycin...

  9. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ... Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B....retroviruses have been developed into some of the most widely used tools in molecular biology. “Retroviruses”...immune system directly). It may seem odder when you consider that out of the 6 x 106 virus-like particles that...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...clock to the 6 o’clock positions at the left-hand side. These steps are common between natural infection...

  10. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    Published
    Dec. 2, 2014, 7:23 p.m.
    ... of regulation of genome functions, we need to identify molecules associated with specific genomic regions... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic... retroviral vectors expressing tagged dCas9 and guide RNA. The retroviral enChIP system enables us to ...system will allow researchers to use enChIP in a wide variety of biological systems. We are currently ...addition, we have recently developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) method ...shipping. Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids...community is a kind of duty for researchers when considering the peer-review system of journals and grant ...

  11. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    Published
    April 23, 2014, 1:08 p.m.
    ... While not ideal, transient expression of viral constructs is a useful tool as it provides a means to ...the specific target cell or tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...of lentiviral vectors in producer cell lines. Identifying less toxic envelope proteins or envelope proteins...to produce viral particles. The components are divided as follows: Transfer vectors contain minimal cis-acting...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...to lower the risk. Biosafety should always be considered with respect to the specific experiments being...

  12. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    ... understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification... abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly 100% in archaea...transposases, by leaving out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar...Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with...with BioRender.com.   Type III Type III is considered the most complex of the CRISPR types and is thought...et al., 2021). IV-A and IV-B lack what you may consider to be an integral component of CRISPR — a nuclease...engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision edits, ...

  13. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    Published
    April 11, 2024, 1:15 p.m.
    ...range, identify a few of the features that confer broad host range in plasmids in nature, and provide tips... meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella spp., Serratia...equally. Only a handful of plasmids have been identified as broad host range plasmids. The properties ...are not fully understood, but scientists have identified some of the features that confer broad host range...metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid with only...Porphyromonas gingivalis, Proteus mirabilis, Providencia spp., Pseudomonas spp., Rhizobium leguminosarum... M. xanthus, Proteus rettgeri, P. mirabilis, Providencia stuartii, Pseudomonas spp., Rhizobium leguminosarum...

  14. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    Published
    Oct. 13, 2022, 1:45 p.m.
    ...Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets...unique experiment. Here, we will introduce you to the wide array of Cas9s (many available through Addgene’s...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...residue of a protein or knocking out a very small peptide. In these cases, you have little flexibility as...specific? Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...

  15. Healthcare Consulting: A Door to the Business of Life Sciences

    Type
    Blog Post
    Published
    Oct. 13, 2016, 2:30 p.m.
    ...accurately communicating ideas in a content-rich, easy-to-understand set of PowerPoint slides. What are some of...learned as a healthcare consultant and give you an idea of what a career in this field involves, and why...a defined path forward. Communication is key as ideas must be relayed quickly both internally and externally..., where case managers are encouraged to use the wider firm’s resources when their teams are facing a crunch...approach brings to patients, doctors, and insurance providers, and is crucial to how our clients’ products will...you’ll have the opportunity to learn the business side of consulting cases, while feeling like you are ...to get your foot in the door When I was first considering preparing for and transitioning to a career in...

  16. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    Published
    April 16, 2020, 1 p.m.
    ...UnLOCKING Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase (Abudayyeh et al 2016...-related single nucleotide polymorphisms (SNPs) from human saliva. SHERLOCKv2 provides a method to detect... expensive, time-consuming, and complex to use outside of standard laboratories. In the case of the COVID... 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave ssRNA or mRNA. It also exhibits a...based on DETECTR technology with the mission to “provide a CRISPR-based platform on which an infinite number...2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573... Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell...

  17. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    Published
    July 23, 2019, 12:46 p.m.
    ...searched for this unique sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor...library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...then screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection...hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A or other Ly6 gene family members ...susceptibility. The Deverman and Wilson Labs independently identified LY6A as a receptor for PHP.B by looking for a...total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive to PHP.B...the PHP.B permissive phenotype of mouse strains beside C57BL/6J and BALB/cJ, and the effects of overexpressed...

  18. CRISPR 101: Drug discovery and CRISPR

    Type
    Blog Post
    Published
    May 4, 2023, 1 p.m.
    ... let’s say you identified gene target X for BRCA-deficient breast cancer and identified several candidate... of two approaches. The first route involves identifying a gene/pathway to develop a drug for (aka a druggable...to their target, or their direct target can be identified if it isn’t known. If a strong candidate emerges...put into them in those later stages. Target identification Not all diseases are fully characterized or...simple pathology. In these cases, preliminary identification of potential targets by CRISPR can accelerate... activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...off target effects, both of which make it a non-ideal drug. If a drug selectively kills the wildtype line...

  19. Plasmids 101: Gene, enhancer, and promoter trapping

    Type
    Blog Post
    Published
    May 2, 2023, 1 p.m.
    ...need to be inside of a gene’s open reading frame, in fact, they can be activated a considerable distance... gene expression are all factors to consider. Once you’ve decided on the type of trap, there are many...world, approximately 50% of predicted genes have unidentified roles as well (Vanni, et al). Is it true that...genes are truly dispensable or are the current identification methods missing something? Here we will review...review some time-tested gene identification systems that may have a leg up on their modern counterparts...just yet. Gene and promoter traps unambiguously identify the gene that they are monitoring as they are ...distance away from a gene which can make identification of the gene itself difficult. The best trap depends ...

  20. Plasmids 101: Walkthrough of Addgene’s Snapgene-Powered Quality Control Process

    Type
    Blog Post
    Published
    Feb. 1, 2018, 3:07 p.m.
    ...we can confirm the identity of the gene and check for any mutations. Once we identify the gene, we can ...talked about how our new Snapgene generated maps provide improved feature detection with an easy to use ...SnapGene for plasmid quality control are: To identify important plasmid features To align NGS sequencing...experiments for further plasmid verification Identifying plasmid features While our quality control process...in Snapgene’s extensive database. Snapgene also provides easy visualization of open reading frames, which...similar features and it allows you to more quickly identify the same feature in other plasmids. Aligning ...Aligning sequences with Snapgene    Once we’ve identified the features within a plasmid, we next verify that...
Showing: 261 - 280 of 895 results