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We narrowed to 987 results for: des

Showing: 181 - 200 of 987 results

  1. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ... friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “...assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. ... Both Snapgene and NEB have tools that help you design primers for PCR amplification of fragments to incorporate...demonstration of how to use Snapgene’s program to design primers for Gibson Assembly. For a simple example...without scars as depicted below. First, you need to design primers to amplify the two fragments while also...the process works best with fragments over 200 nucleotides. This may be because the exonuclease could chew... through an entire fragment shorter than 200 nucleotides before the annealing and polymerization steps...

  2. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...knock-in. Another good design tool can be found at Desktop Genetics .For ssODN design, remember to incorporate... systems, you will have to design your crRNAs using other web-based design tools (for C-terminus tagging... not work in pull-down assays: Epitope tagging provides a solution Proteins - the workhorses of the cell...to mention frustrating. One elegant solution to sidestep the antibody problem is to tag the protein of ...sequences at the intended target site. With the widespread adoption of CRISPR/Cas9 technology and rapid ...bioinformatics tool for guide RNA and donor DNA design, and implemented our optimised protocols into a...genome browser: https://www.ensembl.org/index.html Design crRNA/ssODN: Using Ensembl Transcript ID(s) as ...

  3. High Complexity Golden Gate Assembly with NEB’s DAD

    Type
    Blog Post
    ...rules of thumb for designing your overhang sets. But are those rules the best way to design GGA overhang sets...overhangs with the same three nucleotides in a row; (4) no more than two nucleotides in the same position; (...data-optimized assembly design (DAD) to build out three tools to help researchers design high-fidelity Golden...the traditional rules of thumb for overhang set design allowed for high-fidelity assembly for sets of ... overhangs that violate some of the traditional design rules) for successful high-complexity Golden Gate...with a high potential for mismatches, so you can redesign individual pieces as needed to improve fidelity... a stochastic search algorithm rather than pre-designed sets, so identical searches can return multiple...

  4. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used...primers that bind at either side of the undesired portion, designed to amplify the rest of the vector sequence...primary method to produce the linear fragments, as desired modifications and homologous regions can be encoded...specifically cuts methylated DNA, so it will selectively destroy the template, but not newly synthesized sequences...mutagenesis to complex assembly of multiple genes. Primer design for each type of modification is shown in Figure... main requirement for IVA cloning is carefully designed primers. The majority of mistakes are due to incorrect...homologous sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature...

  5. Plasmids 101: Colony PCR

    Type
    Blog Post
    ...step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers... second option is to design backbone-specific primers. These primers are designed to anneal to sites that...less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert; ...things to consider when performing colony PCR.   Designing colony PCR primers The first and perhaps most ...-specific primers: Insert-specific primers are designed to anneal to an insert-specific sequence. This...backbone but that contain different inserts. When you design primers to anneal outside the cloning site, it ...about insert orientation, then you might consider designing orientation-specific primers. Blunt end cloning...

  6. CRISPR Challenges: Standardization and Homology Directed Repair

    Type
    Blog Post
    ...working with CRISPR than just designing a repair template that contains your desired edit, and I want to make...say about CRISPOR, a tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports...The blog post I mentioned, John Doench’s How to Design your gRNA for CRISPR Genome Editing, gets over ...these numbers show that this basic concept of gRNA design is still challenging for researchers using CRISPR..., and that standardizing and improving the gRNA design process and rules for various applications is necessary...tackle the challenges of DNA repair (with bonus gRNA design resources.) Subscribe to CRISPR updates on our ...in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and Dylan Kwart...

  7. Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

    Type
    Blog Post
    ...DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the RNA guides employed...24 base DNA guides may be more user-friendly, since they can be ordered as oligonucleotides and should ...gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA guides must ...important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also...cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts et al. were unable to show...NgAgo preferred certain guide sequences - using 5 guides/gene for 10 genes, they did not observe differences...a different approach, randomly removing 1-20 nucleotides from the cleavage site specified by the gDNA....

  8. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    ...for CRISPRi are described here: http://www.addgene.org/crispr/qi/ Plasmids described in  Zalatan et ...stepwise protocol on designing and using RNA scaffolds: Delebecque, C. J. et al. Designing and using RNA scaffolds...regulators allows a greater degree of control of desired expression levels dependent upon the type of transcriptional...transcriptional regulator or the number of designed scaffold binding sites. With hundreds of characterized...tunability of transcription is possible. From a design perspective, this is appealing since the RNA scaffold...pairing, allowing for rational, straightforward design. Since the technique relies on base pairing, it...and multidirectional multiplex control via scRNA design is readily apparent in metabolic engineering. The...

  9. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...shipping. However, there were issues that persisted despite our best efforts. With a little over half of our...packages navigated customs and arrived at their destination in a timely manner and at the correct temperature...for their experiment.     Table 1: Most popular guides, protocols, and collections of 2023. Educational...Educational Resource Top Five by Views Guides CRISPR Guide Lentiviral Guide AAV Guide Optogenetics...Electrophoresis Bacterial Transformation How to Design a Primer Creating Bacterial Glycerol Stocks Inoculating...Origin of Replication What is a Plasmid? How to Design Your gRNA for CRISPR Editing The Promoter Region...Analysis Videos What is a Plasmid? How to Design Primers for PCR Agarose Gel Electrophoresis Inoculating...

  10. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...your genome edits, this paper provides detailed instructions on how to design such assays, in addition to...great deal of precision. This means digital PCR provides an absolute measure of target abundance, does ...what kind of assays can be used to sniff out your desired mutations. The assays you will use aren’t that ...often used in real time PCR. These assays can be designed to detect either a donor sequence integrated through...non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation of NHEJ to generate...the target locus, as well as a reference probe designed to bind at an unedited site distal tothe predicted...double strand break, and a NHEJ/ “drop-off” probe designed to bind directly at the predicted double strand...

  11. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    ...essentially any DNA sequence desired. This technology allows you to create a pair of designer proteins that will...nucleases that will bind desired sequences. TALENs are much more reliable. If you design them for a particular... simple to design TALENs. It only takes two to three days from the moment you are designing a TALEN on...Cell, Musunuru, Chad Cowan and their colleagues describe a much more efficient tool for doing that disease...people unfamiliar with this new TALEN kit, can you describe it for us? Musunuru: TALENs are a type of genome...target a particular DNA sequence? Musunuru: The kit includes 832 plasmids containing prefabricated pieces that...to build a DNA-binding domain to recognize any desired 15 base pair sequence in the genome in rapid fashion...

  12. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...screening by PCR may be the way to go. Design a PCR to flank your desired edit site and run the PCR product...nucleotide along with your desired edit. This silent mutation should be designed solely for restriction enzyme... updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells... your cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed...know a few things about your target cells and the desired edits. First, you will need to know what the ploidy...knockout mutations When a knockout cell line is desired, any mutation that introduces a frameshift in the...frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs...

  13. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    ... later this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor ...with each cell line. The editing strategy and design described above can be used to create similar donor ...GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone...plasmid to prevent the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection. Making the...use electroporation, a technique that briefly destabilizes the cell’s outer membrane and allows the components...Addgene’s CRISPR-based Protein Tagging collection includes systems for tagging mammalian cell lines, Drosophila...

  14. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ...and should be designed accordingly. If you are trying activate a gene, you should design a control that...carried out by scientists every day. Although the designs and outcomes may vary, one thing should be present...these control plasmids are critical for the correct design of the experiment.  The Experiment: Knockdown the...control plasmids Part of planning your experiment includes determining what factors need to be controlled...microscopy images resulting from our experiment that includes the aforementioned internal control and is consistent...correct interpretation of the results because it provides an important reference point when analyzing the...positive control, the internal control vector, was described earlier. Once you are sure your conditions are...

  15. Michael J Fox Foundation (MJFF) Plasmid Collection

    Type
    Collection
    ... Neurodegeneration Research Collection , which includes other areas of this research topic. Michael J....Browse or search the table below. ID Plasmid Description Gene/Insert Industry PI Return to top...

  16. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...overall HDR rates, we have shown recently that desired genome-editing events by HDR can be generated more...genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we were very excited...introduced. Although we were able to introduce the desired mutations in genes associated with dementia, such...CRISPR from re-cutting the target sequence once the desired edit has been introduced. We found that blocking...only saw incorporation of the blocking mutation. Despite the fact that both the blocking mutation and our...thinking further about the distance relationship described above, we realized that, in addition to increasing...efficiency a guide RNA targeting a cut <10 bp from the desired mutation should be used. For a heterozygous event...

  17. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...different ways. We describe a number of issues with some of the experimental designs used to make these...of Amsterdam. The previous post in this series described a practical approach to selecting a bright fluorescent... protein. Photobleaching is the irreversible destruction of a fluorophore under the influence of light...photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable...photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent...large number of emitted photons before they are destroyed. Factors that affect photostability The photobleaching...fluorescence, which, in its simplest form, can be described by a mono-exponential decay (analogous to radioactive...

  18. Synthetic Biology - Cloning and Genomic Tools

    Type
    Collection
    ...available from this depositor's lab. Plasmid Description Gene/Insert Vector Type PI Publication Back to... plasmids from this depositor's lab. Plasmid Description Vector Type Selectable Marker PI Publication ...

  19. Depositor Collections

    Type
    Collection
    ...'s repository contains 161,184 plasmids, which includes plasmid collections from depositors. To help you...Dehydrogenase CUREs Community (MCC) Collection Pleiades Promoter Project: mini-promoters to drive selective...

  20. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...SapTrap, the user first designs either oligos or synthetic DNA for the desired gRNA target sequence, as...specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...need for PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site...accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library containing several... and a Cas9 expression plasmid will insert the desired genetic tag and marker sequence into the targeted... a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple target... not work with C. elegans, several groups have designed modular toolkits to assist with tagging genomic...
Showing: 181 - 200 of 987 results