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We narrowed to 667 results for: FIE

Showing: 81 - 100 of 667 results

  1. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ...this case, regions of gene S and Orf1ab gene are amplified. RPA reactions also contain reverse transcriptase...transcription and Cas13 is then used to detect the amplified region of interest. Cas13 uses a complementary...resulting in fluorescence. Cas12a binds all the amplified target regions and results in the continuous cleavage...subject to reverse transcription. This is then amplified by PCR using Taq Polymerase and then transcribed...Highlights: Reverse-transcribed RNA samples are amplified by PCR instead of RPA. Unlike RPA, this technique...typical PCR machines, the authors use affordable, field-ready, Bluetooth-enabled PCR machines that can even...made accessible by the DIY-Bio movement. Rapid, field-deployable nucleobase detection and identification...

  2. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified fairly easily... the desired mutation) in a PCR protocol that amplifies the entire plasmid template. The parent template...obtained with PCR screening using the primers specified by the arrowheads in (A). Experimental guidelines...Methylation deficient bacterial strains can be identified by the dam13(-) mutation – you’ll want to steer...mutagenesis. Nuclease To remove the template DNA (unmodified plasmid) a restriction digest with DpnI is used...the bands present in the digest products of the unmodified plasmid will be split into two smaller bands ... two smaller bands visible in a digest of the unmodified plasmid into a larger band (not shown). As a ...

  3. A Look at Addgene's QC Process

    Type
    Blog Post
    ...different than what was originally intended. Sharing unverified plasmids can lead to far reaching consequences...sequences and/or features. Most of these regions are identified during the QC process, following sequencing and...confidence. If this approach does not resolve the identified differences, the plasmids are sent out to Plasmidsaurus...Illumina MiSeq.  Resolving QC issues If the QC team identifies a QC issue, they will contact the depositing ...depositing lab to review the discrepancies. Often, the identified issues will not affect function and the plasmid...page to make requesting scientists aware of the identified discrepancy in the plasmid, and that it is not...function, we request the lab provide a new, sequence-verified replacement for the plasmid, and the full QC process...

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

    Type
    Blog Post
    ...regions that are bound to modified proteins called histones. We can detect modified histones with “GPS for...fractal). Enhancer biology is a complex and dynamic field. We’re going to focus on a tried and true method...DNA’s 3D geometry and the chemical properties of modified histones, a genomic region that has mono-methylated...genome math, we’ll need a bit of shorthand from the field of epigenomics: H3 – one of NANOG’s associated ...genetically programmed to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population...enhancers, promoters, insulators, and more. I then identified valid gRNA sequences without off-target effects...terminator), and no off-target effects for the identified 23-mer gRNA. Off-target determination was established...

  5. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...library, followed by DNA isolation. The amplified library is verified through NGS sequencing to confirm library...Producing Your Lentiviral Library Once you've amplified–and verified!–your pooled library DNA, the next step...you’ll need to develop your own protocol that amplifies without disrupting plasmid representation. A good...backbone.  Verification Once your library has been amplified, you'll need to verify your library by next-generation... sequencing (NGS). The representation of your amplified library should be very similar to that of the ...libraries, NGS is the best way to validate your amplified library and verify that you have not skewed the...

  6. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    ...the CRISPR toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a ...cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov et al., 2015). They were using...the bacterial genome. From this analysis, they identified 53 potential candidate genes that fell into 3...recognized and cleaved its target RNA sequence as specified by the crRNA sequence, it adopts an enzymatically...nucleotides containing a sequence of interest (red) is amplified using Recombinase Polymerase Amplification (RPA...Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides are combined with Cas 13a in complex...enzymes continue to make a splash in the CRISPR field. We feel that the culture of sharing within the ...

  7. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...signal from this reporter can be measured and quantified to determine the presence and quantity of DNA...Enzymatic Reporter UnLOCKING Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase...assay, targeted DNA or RNA from a sample is first amplified using RPA (recombinase polymerase amplification...RPA is coupled with T7 transcription to convert amplified DNA to RNA for subsequent detection by Cas13. ...used easily and effectively in the lab and in the field (Myhrvold et al., 2018). Now, the Zhang lab has ... SARS-CoV-2 RNA. The test is started using RNA purified from patient samples and can be read using a dipstick...reaction. To enhance sensitivity, the DNA is first amplified through isothermal amplification by RPA. When ...

  8. Tips for Getting a Faculty Position

    Type
    Blog Post
    ...completing the research will advance the field, create a new field, impact disease treatment, etc., again...that search committee members are experts in your field. Not true. Even if you are applying to a department...possibly the Dean, and even some people in your field that still need to understand your specific questions...display your deep knowledge of your experiments and field. It's much more impressive to answer an expert's...DO NOT need to present a detailed review of your field or the finer points of your techniques. You DO need...need to be prepared to know the literature of your field and all of the caveats and alternatives for an approach...correct positions. What is the knowledge gap in the field? How will your approach resolve this problem? (2...

  9. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...approximately one-third of cellular proteins are modified through phosphorylation (1). However, the expansion...databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events ...necessary for study. Moreover, the ability to isolate purified phosphorylated products usually depends on tools... vitro. The establishment of a system in which purified, recombinant protein could be produced with site-specific...necessary for phosphoserine incorporation is a modified bacterial strain, C321.ΔA. Because the phosphorserine...the TAG amber stop codon, using the OTS in an unmodified bacterial strain would result in read-through... of interest. Two expression systems have been verified for use by the Rinehart lab: pCRT7 NT TOPO E17TAG...

  10. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...1: Guidelines for the data fields in the deposit spreadsheet Data Field Description Plasmid ... plasmid and will help a scientist outside your field understand if this plasmid will work for them. ...synthetic biology, affinity reagent/antibody, unspecified, or other. If other, indicate type in “Vector...other, indicate strain in the “Growth Strain Other” field, and contact Addgene. We may require that you send...explanation in the adjacent Patent/License Explanation field. Filling out and sending the deposit spreadsheet... and a pop-up instruction box for the “Purpose” field. If you ever have a question about what should go...

  11. Using a Light Microscope Protocol

    Type
    Protocol
    ...lens which magnifies the image, then the ocular lens, where the image is further magnified before it finally...4x objective magnifies the image four times the actual size, the 10x objective magnifies it 10 times, ...also 10x, you would actually be seeing a 100x magnified image of that object! Equipment Light Microscope...aperture are in focus. Center the aperture in your field of view and then slowly open the diaphragm until...the stage (and ultimately your slide) until your field of view is centered on a region of interest. If ...

  12. Plasmids 101: Protein Expression

    Type
    Blog Post
    ... structure of proteins. Most proteins are then modified via an array of post-translational modifications...interactions and functions. Proteins can also be purified for use in molecular biology research (eg. polymerases...polymerases and other enzymes might be purified and used to manipulate DNA), or in medicine (e.g. insulin...translate and modify your protein of interest. Modified proteins can then be extracted from lysed cells... interest, and then lysed. Protein can then be purified from the cellular debris. There are several popular...systems, proteins are assembled in vitro using purified components of the transcription and translation... ability to assemble proteins with labelled or modified amino acids that are useful in different downstream...

  13. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    .... Once you are satisfied with the plasmid information, click on the blue "Save Verified Data and Return... to submit published or unpublished plasmid(s). Fields marked with an asterisk are required. If submitting... hundred to millions. Pooled libraries must be verified by next-generation sequencing. Please see the ... theoretical — it does not have to be entirely verified by sequencing. Upload any support files that will...saved and the page will open a new set of entry fields to describe the next insert. Repeat as needed for...Bacterial Expression vector. If your plasmid can be classified in multiple categories, please check all of the... that the material I am submitting to Addgene qualifies as BL1 or BL2 as defined by NIH guidelines. I ...

  14. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...discoveries in various biological fields. In 2015, the Fujii Lab identified non-coding RNAs associated with...adaptations of this system have been made in different fields of study. In addition to the identification of ...incubated with fragmented chromatin or library of purified DNA for purification of a specific chromatin or...Biotinylated dCas9, with bound DNA, can then be purified by streptavidin high-affinity purification. This... malignancies. Additionally, the Tapscott Lab identified regulators of DUX4, a transcriptional regulator...CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator of IGF2 imprinting...

  15. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...necessary to remove iodixanol prior to use of your purified virus. Nonetheless, we recommend performing a ...a buffer exchange before using the purified AAV in vivo . The iodixanol gradient in this protocol is composed...genome containing) particles. On average, iodixanol-purified AAV preparations contain ~20% empty particles....60% iodixanol step Carefully add up to 5 mL of clarified supernatant on top of the gradient. Use 1X PBS...fractions obtained from the 40% phase contain the purified AAV. Option #3 Puncture the QuickSeal tube slightly...vertical black line indicates the location of the purified AAV. Left image adapted from Zolotukhin, S., et...the position of the needle for harvesting of the purified AAV using option #1. Right image adapted by permission...

  16. Tips for Writing a Good Cover Letter

    Type
    Blog Post
    ...you stand out.  In some fields, a cover letter is vital, particularly in fields where communication and...and writing are key skills. Other fields, often highly technical or skills-based ones, may not use them...process, it's a good idea to talk to people in the field to understand what the norms are. What is a cover.... They can be skills-based: if you're switching fields, you can use your cover letter to explain why your...scientist with over [number] years of experience in [field of research], and have excellent [list the most ...that stage, cover letters can be a barrier for qualified applicants. In short, while a cover letter can...

  17. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    ...previously identified essential genes while previous Cas9 and shRNA library screens only identified  60% of...a 10% false discovery rate and identified 67% of previously identified ricin toxicity genes. The screen...non-essential genes for both pathways had previously been identified (Hart et al, Bassik et al). The library performed...essential for growth and would not be expected to be identified in a CRISPR knockout screen. Improving the Design...difference in depletion of ricin sensitizer genes identified with short and long guides. Together these results...

  18. Viral Production at Addgene

    Type
    Blog Post
    .... Figure 1: Silver staining of purified and non-purified AAV subjected to gel electrophoresis. ...adenoviral helper sequences. AAV preparations are purified by iodixanol gradient ultracentrifugation and ...and an internal control of known titer (protocol modified from Lock et al., 2014.) Previous experience suggests...within representative vector preparations was quantified with electron microscopy after negative staining...staining. Empty vector particles can be identified after negative staining and appear darker than full vector...droplet digital PCR (ddPCR) titration protocol (modified from Wang et al., 2018). Briefly, 293T cells are...

  19. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...Microsoft Excel or similar software Reagents Purified antigen Purified recombinant antibody Isotype control antibody...describes how to run an indirect ELISA against a purified antigen to demonstrate the expected antibody dose...Enzyme-Linked ImmunoSorbent Assay (ELISA) against a purified antigen to validate an antibody. This protocol...the Antigen Standard and coat the plate Dilute purified antigen to 20 ng/µL in a microfuge tube and vortex...concentration. Prepare serial dilutions of the purified antigen as follows: 2 ng/µL : Add 100 µL of 20...coated with serial dilutions of human recombinant purified Desmin (blue) or a negative control protein, Apolipoprotein...

  20. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    ...studies. Additionally, Spinach, the first aptamer identified to bind DFHBI, is highly unstable in vivo due...site-directed mutagenesis of Spinach. In doing so, they identified the nucleotides that contribute to the instability...Broccoli, the other green fluorescent aptamer, was identified through directed evolution. First, a pool of ... that activate the fluorescence of DFHBI were identified using systematic evolution of ligands by exponential...hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified from DsRed. DFHO shares many structural properties...Mango, coined Mango II, III, and IV, were then identified through microfluidic-assisted in vitro compartmentalisation...possibility for scientific advancement, and in a field with as much potential for growth as RNA biology...
Showing: 81 - 100 of 667 results