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We narrowed to 704 results for: des.1

Showing: 141 - 160 of 704 results

  1. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,...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...

  2. Plasmids 101: Gateway Cloning

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    Blog Post
    ...pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible pLIX_403. The chosen attR destination vector will... our desired constructs: lentiviral expression of the human KRAS gene in mammalian cells. STEP 1: Generate...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR sites...regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. ...the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction... Find Gateway cloning plasmids here! References 1. Chee JY, Chin CF (2015) Gateway Cloning Technology...clone in their desired DNA sequences. Gateway technology relies on the two reactions described below: The ...

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

    Type
    Blog Post
    ...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...rules of thumb for designing your overhang sets. But are those rules the best way to design GGA overhang sets...

  4. Plasmids 101: Control Plasmids

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    Blog Post
    ... that includes the aforementioned internal control and is consistent with the result in Figure 1 could...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...scientific inquiry and investigation. References: 1. The problem of pseudoreplication in neuroscientific...and should be designed accordingly. If you are trying activate a gene, you should design a control that...

  5. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    ...to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI digestion... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894.... Traditional restriction cloning is one of the oldest of these techniques and is a multi-step process...

  6. CRISPR Antimicrobials

    Type
    Blog Post
    ..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...beta-lactam resistant population to the antibiotic, despite this resistance gene being encoded by a high-copy...currently undergoing a renaissance. Yosef et al. designed a two-phage CRISPR system to resensitize antibiotic-resistant...

  7. Quick Guide to All Things Lentivirus

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    Blog Post
    ...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...internal promoter, and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral...to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one...plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "Redesign of retrovirus packaging...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...

  8. 5 Uncommon Ways to Gain Career Skills During an Uncommon Time

    Type
    Blog Post
    ...need a job. Start now. Informational interviews are 1:1 discussions with someone who has interesting experiences...conversation builds a relationship for the future and the 1:1 format is good for everyone including those that ...out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group We don’t all get the...harder to build relationships when you are more desperate or needy so you can’t wait until you imminently...

  9. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ...the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers (no need to add ... strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different DNA strand...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...enzyme and Taq, with Taq used in an excess ratio of 10:1. Gel purify your PCR product and incubate it with ...you to see colonies within 8 hours. References: 1. Shuman S. "Recombination mediated by vaccinia virus...

  10. Getting to Know Expansion Microscopy

    Type
    Blog Post
    ...protocols. A typical ExM protocol (Fig. 1) follows this structure: Step 1: fix (and maybe label) your sample...microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1  Mikhaylova M, Cloin BMC, ...just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify ...16:71–74. https://doi.org/10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023...Microscopy. What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem....improve label density (Mikhaylova, et al. 2015).  Despite these limitations, ExM is a valuable tool, especially...Bournonville L, et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria ...

  11. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    ...Jacqueline N Crawley  [et al]. 2012;Chapter 1:Unit 1 20 1-18. doi: 10.1002/0471142301.ns0120s59. PMID:...mutually exclusive) commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. ...can be broken down broadly into three main steps: 1) Selection and expression of your opsin of choice ...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...follow him on Twitter @derekpsimonphd.    References 1. Tye KM, Deisseroth K. Optogenetic investigation of..., Jacqueline N Crawley  [et al]. 2013;4(435):4 35 1-4  20. doi: 10.1002/0471142301.ns0435s65. PMID: 25429312...

  12. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...is right for their experiment.     Table 1: Most popular guides, protocols, and collections of 2023. ...our repository to an amazing 111 countries (Figure 1)! But while we were making the materials available...accessibility varied country to country.   Figure 1: Number of countries Addgene distributes to per year...date, we have partnered with six distributors (Table 1), in China, Korea, Japan, Brazil, India, and Mexico...in 2023, from visitors in 218 countries. In Table 1, you can see our most popular educational resources... execute a step can provide needed clarity (Table 1). Our most popular video, however, is “What is a Plasmid...shipping. However, there were issues that persisted despite our best efforts. With a little over half of our...

  13. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    ...coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003):...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site will...genOway press release for more information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O...switches? FLEx (or “flip-excision”) switches were designed as a genetic tool for researchers to conditionally...does the FLEx switch work? Let’s say you want to design a genetic FLEx switch that turns BFP expression...

  14. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ...Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination vectors... Let's walk through designing a CRISPR vector the Fragmid way!    Figure 1: Selecting your vector...answers in step 1, so you’ll only be able to select components that work with your design. Note that you...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden...website, which breaks the design process down into four easy steps, provides the real value for the user...potential of them not working, or spend a few weeks designing and cloning the vectors you actually want. But... Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach to cloning...

  15. Antibodies 101: Producing Recombinant Antibodies

    Type
    Blog Post
    ...materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created ...EBNA1 Cells. Methods Mol Biol. 2018;1850:1-16. doi: 10.1007/978-1-4939-8730-6_1. PMID: 30242676. Frenzel...Neuroscience Research. Curr Protoc Neurosci. 2020 Dec;94(1):e106. doi: 10.1002/cpns.106. PMID: 33151027; PMCID...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...case, a buffer exchange can be performed using a desalting column or an ultrafiltration concentrator. As ...suggests, you may also concentrate your prep to the desired concentration using these columns. If you are conjugating...

  16. Multiplex Genome Editing with CRISPR-Cpf1

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    Blog Post
    ...For these experiments, cells were infected with a 1:1 ratio of two AAVs,  one expressing Cpf1 and the other...comparison of Cpf1 vs. Cas9, see the table below. Table 1. Comparing the Cas9 and Cpf1 CRISPR Nucleases   ...Overall, these approaches have two main drawbacks: 1) Most rely on transfection of more than one vector...leave them in the comments below.        References 1. Zetsche et al. "Multiplex Gene Editing by CRISPR–...editing!  A recent paper from Feng Zhang's lab describes how to use Cpf1 for multiplex genome editing.  ...sequence NGG TTTV PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing....  Similar to a jigsaw puzzle, the oligos were designed with sticky ends that only anneal together in ...

  17. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

    Type
    Blog Post
    ... process. Also refer to Figure 1 for a visual overview! Figure 1: General overview of the MoClo...part from an Addgene MoClo kit, to construct a Level 1 vector with a promoter and terminator flanking the...acceptor (bottom) for CDS1 parts. (B) Example Level 1 assembly. The choice between different Level 0 vectors...properly. Do this sequentially for your Level 0, Level 1, and Level 2 assemblies using the plasmid sequence...after each transformation. This could be as few as 1–2 colonies if you’re an experienced MoClo user, but...manner. These videos can be especially useful when designing new primers to clone your sequence of interest... your cloning. Most sequence analysis software includes Golden Gate cloning tools that simulate the restriction–ligation...

  18. Using AAV for Neuronal Tracing

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    Blog Post
    ...but also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011...Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 Table 1: Common AAV Serotypes and their target tissues (adapted... serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans et al., 2007; Howard et...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...Wolfe, J.H. (2014). Adeno-associated virus serotypes 1, 8 and 9 share conserved mechanisms for anterograde...,9 in rat neurons and glia in vitro. Virology 372(1), 24–34. PubMed PMID: 18035387. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...

  19. How-to: 5 Steps to a Great Panel Discussion

    Type
    Blog Post
    ...not let the panelists introduce themselves. I do a 1-2 minute introduction. If you chose good panelists...take 5 minutes to introduce themselves and, voila, 1/2 of your time is gone with no interesting discussion...introductions. The moderator asks questions, calling on 1-2 panelist for each question in a way that elicits... time you are at a talk and the speaker goes over 1 hour, watch what happens to the attention of the audience...postures shift, they are checking out. If you can, plant 1-2 good questions with trusted audience members. A ... asking the panelists not to use any slides for the event. Slides are for presentations, not discussions...discussions. I will rarely work with a panel if slides are allowed. Don't put your panelists behind a table. ...

  20. Which Fluorescence Microscopy Technique is Best for Me?

    Type
    Blog Post
    ... in modern biology (1). When performed correctly, fluorescence microscopy provides a high contrast image...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished... the resolution of a light microscope (see Figure 1). What microscopy technique should I use for my experiment...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample...of modern day fluorescence microscopes (see Table 1). Thin dynamic samples Ex: monolayers of live cells...excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon has been a very slow imaging...manufacturers have similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy...
Showing: 141 - 160 of 704 results