Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of the Addgene plasmid sequence database.

Information

In Category

We narrowed to 1,010 results for: ple

Showing: 221 - 240 of 1010 results

  1. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ... weeks to a couple of months and requires that all of the experiments have been completed. Therefore, ...opportunities in academia - the main reason most people enter postdoctoral training. Similarly, there are...essays in the journals Nature and Science, for example). This blog post is about how to get the most ...a Group Leader position, what will you need? Multiple first or corresponding author publications (published... questions like: What figures are missing? For example, are additional controls necessary? As you build...interesting questions in your own lab. 2. Seek multiple mentors during your postdoc At the end of grad...Discussions can reveal mutual scientific interests, complementary perspectives, a shared communication style. ...

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

    Type
    Blog Post
    ...remaining letters should be from independent people – i.e. people who have not worked with you directly or... connect with people who could be beneficial during a career search. Doing these simple things will make...refine and build skills overtime for success. Many people will have different roles, titles, and responsibilities... to find the perfect job immediately after you complete your Ph.D. or postdoc. Foreign national researchers...standard of proving that you are one of the top people in your field. No employer is necessary and no ...to the field that have had a major impact. For example, you were the first person to identify a novel ...successes in the performing arts- not relevant at all. Please do not include the fact that you are a classically...

  3. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...assembly, assembling fragments in multiple steps to avoid using multiple fragments that share homology in...overhangs perfectly match up. They just need enough complementary ssDNA to hold the plasmid together, even if ...if there are gaps or flaps remaining. This incomplete plasmid looks like a homologous recombination intermediate...to work fixing up the “damage” and producing a complete plasmid. Besides being compatible with any vector...either by annealing a mixture of products or from incomplete PCR — although higher DNA concentrations are ... separate PCR products (middle branch); or by incomplete PCR, which skips the final extension step to ...single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product...

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

    Type
    Blog Post
    ...of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and upon binding, gets activated...the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity ...strip. Highlights: Of the 83 known COVID-positive samples tested, DETECTR has a positive prediction rate ...°C water bath Approach: RNA is extracted from a sample and subject to isothermal recombinase polymerase...presence of two or more mismatches in the cRNA:target duplex and will not give a positive result for highly ... can be performed in two steps for challenging samples as in Two-Step-SHERLOCK or in a fast, high-throughput...incubated at one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the...

  5. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...mutational scanning to simultaneously test how multiple amino acid changes affect a protein of interest...and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational...a selection system. You can test the effect of multiple mutations in combination and possibly discover...in protein activity. See Natarajan et al for an example of this. Plasmid library generation prior to one...mutations are introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil...second universal primer is used to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled...uracils that are in DNA and ExoIII recognizes nicked duplexed DNA. This results in the final product having ...

  6. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ... exact amount of protein in your sample, you’ll compare your samples to a standard curve. This curve is...the known samples is used to calculate the concentration of protein in the experimental samples. Get more...expression of multiple proteins on the same cell simultaneously as well as compare between samples. Learn more...heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable region ... protein because the antibodies will recognize multiple epitopes on the protein. However, each animal ...optimized for specificity and sensitivity, for example by changing certain amino acids to improve its ...expression in cell lysates, whole cells, or tissue samples. Many experiments that use antibodies use a primary...

  7. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...This allows for the multiplexing of sgRNAs against unique genomic targets or multiple sgRNAs against the...enzymes (Weber et al., 2011). It can be performed at multiple levels to generate anything from individual transcription... favorite gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits...assembly can occur. It is quite extensive with a supplemental add-on kit and is compatible with E. coli and...modules, and many more of the usual suspects. The Multiplex Yeast Toolkit builds on the YTK and adds several...capabilities, including conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger...intuitive CRISPR MoClo application is to assemble multiple unique sgRNAs into a multi-TU targeting construct...

  8. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    ...activation experiments. In cases of multiplex gene regulation (activating multiple genes at once), however, SAM... peptide array to fused with multiple copies of VP64. By having multiple copies of VP64 at each loci of...VPR fuses a tripartite complex with dCas9 to activate transcription. This complex consists of the VP64 ... of CRISPR/Cas systems, gene activation across multiple loci was an arduous process. When using zinc finger...sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest. Upon binding, CRISPRa...upon the initial dCas9-VP64 method, so there are multiple options to choose from when looking to activate...CRISPR activator. While it requires a relatively simple construct, it exhibits modest levels of gene activation...

  9. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ...the plasmid complements the deletion). Find the Plasmids Here Plasmid features enable multiple student experiments...abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is corrected,..., well-studied and inexpensive. In addition, a complete set of well-annotated deletion strains is available...occurs through a well-characterized pathway with multiple enzymatic steps (5). In their experiments, students...ura3 mutants and cannot grow without uracil supplementation (6). Expression of MET/Met genes from the pYES2.1...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...

  10. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE (Multiplex Automated.... Adding complexity, they found that they could also include a CRISPRi system for multiplexed modulation...functions in S. pneumoniae and can be used to generate multiple mutations simultaneously (Jiang et al. 2013). ...mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is possible with...pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing...multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria...transcription by physically blocking the elongation complex from binding the DNA or extending the transcript...

  11. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ... The simple answer is that sometimes it is necessary or desirable to control or look at multiple genes...together for complex genetic analysis in Drosophila or for precise control over multiple genes in synthetic...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit...that transcription is now OFF.   We’ll look at examples of both natural and engineered repressible promoters...transcriptional activation domain VP16 taken from the herpes simplex virus. tTA binds to promoters containing TetO elements...in mammalian cells, this system has also been implemented in a variety of other organisms, such as sphingomonads...UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4...

  12. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

    Type
    Blog Post
    ...production site to its final destination. As an example, most secreted proteins are produced in the endoplasmid... the untagged, wild-type protein. You may, for example, validate that the fusion protein localizes properly...FPs to highlight various subcellular structures. Please see Figure 1 for commonly used markers for major...fusions to visualize subcellular structures. For example, you might use your fluoresent protein fused to...and potentially which other proteins it forms complexes with. These “colocalization” studies give insights...within the same subcellular structure or protein complex. By coexpressing both the marker protein and the...possible to determine the location of proteins within complex structures. In order to interpret colocalization...

  13. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors...systems work using a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade...compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade is an important... Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex, developing... settings — mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR...component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination of Cas5, Cas6, .... It was originally suspected that Cas3 moved a couple of kilobases down the strand of DNA, Cascade caught...

  14. Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

    Type
    Blog Post
    ...constructs to allow people to, for example, knock out a gene with simultaneous complementation, with a knockout...enable other people to do that too. We don’t have the capacity to send tools out to people ourselves, but... but we’d really like for people to use them. The MoClo Plant Kit makes it really easy to clone genes ...but have comparable expression levels to things people do use a lot. The problem in plant science is there...similar stability to transcripts as the ones that people use all the time. Now there are many, so if you... those working in plants, including many things people already use. It makes the switch to Golden Gate... Golden Gate is a conceptual barrier. Sometimes people don’t know how to start. They might want to use...

  15. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    ...process such as primer and plasmid design. Examples:  “multiplex design”, “multi-method design”, and “single-method...deals in post-experiment quality control, a good example is CRISPR-GA. These tools assess repair events ...published 1 Jan 2013, no longer available), is a simple catalogue of reported sgRNA designs. The second...similar to EENdb than CrisprGE, but is also a good example of how database tools can be used to aid design...this category are the most common between tools. Examples: “single-target design”, “multi-target design”...considered extremely useful, depending on design goals. Examples:  “feature aware”, “SNP aware”, “secondary structure... common utility functions are batch design or multiplex features. However, tools aiding in primer design...

  16. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ... as the building block of our understanding of complex biological processes. Many tools exist to perturb...in the context of the biological question. For example, while gene knockouts have long been considered...silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and Doudna, 2013). RNAi... translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result, gene... of interfering RNAs.   RNAi vs. CRISPR: Complementary techniques It is fair to say that CRISPR has...of the technique—all that is required dsRNA complementary to your gene or genes of interest to trigger...constructs that enable insertion of ~400 bp complementary to your gene target as inverted repeats (Figure...

  17. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...translational, biomedical, or applied sciences), people conduct research to answer questions about how ...applied. The current pandemic serves as a striking example of the applicability and importance of research...you’ll see in a lab. Position Common tasks Principle investigator (PI) Runs the lab Writes a lot ...questions and your excitement will in turn inspire the people around you. Be proactive Rather than applying only...the best research experiences typically come when people actively seek out science that genuinely excites...experience more tailored to personal interests. Talk to people, ask questions, and dig deep Talk to your professors...into is really important.  As you’re talking to people, though, try to be conscious of who has your best...

  18. Viral Production at Addgene

    Type
    Blog Post
    ...single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR. Human... lot. At Addgene, AAV particles are titered by droplet digital PCR (ddPCR) using primers and probes targeting...specific QC experiments were performed on your lot, please contact us. Full sequencing of the Viral Genome...of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample.   Sterility Viral vector preparations are ...Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified...

  19. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...splitting up a sample into thousands of physically isolated partitions (such as droplets or wells on a ...clones for complete functional knockout of your target gene. Mismatch nuclease assays are completely “blind...of target DNA molecules present in the original sample with a great deal of precision. This means digital...theoretically detect a single target molecule in a sample. Digital PCR assays for mutation screening Now...use aren’t that much different from conventional duplexed primer probe assays often used in real time PCR.... The drop-off probe is designed to perfectly complement the wild type sequence and is not capable of ...amount of both mutated and wild type target in each sample can be quickly calculated (details in [2]) and ...

  20. Flow Cytometry Readouts: Yes, No, and Everything in Between

    Type
    Blog Post
    ... Figure 1: Example of yes-no flow plot of the (negative) control (left) or with the sample (right). Negative...designed your first flow panel, you’ll load your samples into the cytometer and see one of two results for...protein, a yes-no plot is the best-case result. For example, you have introduced a GFP protein into your cells...somewhere between a 104–105 signal intensity. If your samples and controls were processed the same way, the GFP...Negative and positive samples are clearly distinguishable as two separate populations. Created with biorender.com...biorender.com. Pro tip! If your samples and control underwent different treatment processes, you may...the one shown in Figure 1.  Figure 3: An example of a histogram plot for a yes/no flow cytometry...
Showing: 221 - 240 of 1010 results