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 795 results for: Tec

Showing: 141 - 160 of 795 results

  1. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ... a revolution in genome engineering, with new techniques rapidly being developed. Performing a CRISPR ...potential for off-target effects. Cas9 RNPs are detectable at high levels shortly after transfection, and...plasmid based methods. One of the most common techniques for delivery of RNPs is electroporation (A in...the cytoplasm. In addition to the use of this technique in cell culture, it has also been applied to genome...combined with cell-type specific reagents in a technique known as nucleofection, which forms pores in the..., allowing for entry of a DNA template. Other techniques, such as lipid-mediated transfection (B in the...using this method for in vivo applications. This technique utilizes Cas9 proteins harboring receptor ligands...

  2. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    ...Sample preparation for these techniques sometimes makes it more difficult to detect protein tags using antibodies...availability of reliable primary antibodies for their detection, particularly when antibodies to the POI are non-specific...multimeric tags are frequently insufficient for detection when the POI is weakly expressed. As these epitopes...have no intrinsic visual signal, they cannot be detected directly in live imaging experiments. It is therefore... are both intrinsically fluorescent and can be detected with highly epitope-specific, fluorescently-labeled...can be easily expressed in the brain and can be detected through their direct fluorescent signals or using...antibody binding sites and primary antibody-based detection, smFPs create much brighter signals for weakly...

  3. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ... commercially available mycoplasma detection kits that use detector cell lines, ELISA, fluorometry, or...Incidence, sources, effects, detection, elimination, prevention.” Cytotechnology 39.2 (2002). 75-90. Pubmed... lines, or infected personnel and can persist undetected; unlike infections with larger microbes such ...or bacteria, mycoplasma can be extremely hard to detect with levels reaching 108 cells per ml before the...or PCR-based approaches to detect even low levels of contamination. For those labs that prefer a more ...more economical mode of detection, Uphoff et. al. outline a do it yourself PCR-based approach complete with...alters microarray gene expression profiles.” Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3...

  4. Addgene Welcomes John Doench to the Advisory Board

    Type
    Blog Post
    ...consists of prominent members of the bioscience and tech transfer communities who help guide and promote ...that had Platforms with a capital ‘P’ focused on technology ... has been critical to the Broad’s success ...biological questions, you need a tremendous amount of technology development alongside it.” Doench starting working...Perturbation Platform and encompasses a variety of technologies to functionally investigate the human genome...genome. Doench says he enjoys working on technology development under the Platform model at the Broad Institute...Institute because they are “not just developing technology for its own sake,” but work in true collaboration...history of RNAi left him a bit skeptical of the technology at the start of the CRISPR craze, but he says...

  5. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...microscopy technique for your study see Addgene’s blog post Which Fluorescence Microscopy Techniques is Best...fluorochrome is removed from all other detectors except its dedicated detector. To ensure proper compensation ...cell health, analyzing cell cycle stages, and detecting specific populations of cells in a complex tissue...sample (1). When combined with cell sorters, this technology can be used to isolate a specific subset of cells...fluorescence intensity.   What flow cytometers detect Cell size and complexity, measured using visible...directions. The degree of scatter is captured by detectors and provides useful information about the cell...emits a fluorescent signal that is captured by detectors. The strength of the emitted photons varies from...

  6. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...CRISPR 101" post. Next-generation sequencing technologies are the gold standard but they remain cost-prohibitive... assays (e.g. Surveyor® or T7E1) for mutation detection. While these methods paved the way for mutation...introduce digital PCR as an emerging validation technology. Digital PCR has several advantages over mismatch... require standard curves, and can theoretically detect a single target molecule in a sample. Digital ... real time PCR. These assays can be designed to detect either a donor sequence integrated through homology-directed... remainder of this post will focus on assays to detect NHEJ. These assays include a forward and reverse...between 200 and 500 ng of purified PCR product. Low detection limits: Digital PCR assays can accurately quantify...

  7. Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

    Type
    Blog Post
    ...popular technique in neuroscience and developmental biology. This type of cell labeling technique involves...group developed the original iteration of this technology in 2007. Used to label neurons in the cerebellum...cerebellum of mice, the team appropriately named their technology Brainbow. Brainbow 1.0 used the fluorophores ...construct but do not undergo recombination can be detected by immunostaining of the truncated YFP. In Thy1...CMV-Brainbow construct at the embryonic stage. This technique could be used to target Brainbow to neurons by... promoter. Zebrabow is an extremely versatile technology as recombination can be induced in developing...cells. Tetbow optimizes Brainbow for current technologies Tetbow (Sakaguchi et al., 2018) overcame the...

  8. Visualizing Protein Turnover In Situ

    Type
    Blog Post
    ...Einstein College of Medicine has recently published techniques to follow translation in living single cells....cells. However, before CRISPR-Cas9 these techniques were quite tedious and, even here, there is the question... symposium seeking inspiration to resolve the technical hurtle of monitoring protein turnover of specific...interesting talk by Prof. Erin M Schuman, a former Caltech researcher, now Director at the Max Planck Institute...Frankfurt am Main, Germany. She presented a novel technology, back then still unpublished, that allowed the...synthesized proteins with FUNCAT Professor Schuman’s technique applies metabolic labeling using a noncanonical...noncanonical amino acid, azidohomoalanine (AHA). In the technique, AHA is incorporated into newly synthesized proteins...

  9. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...Scientist at the Broad Institute, John Doench. CRISPR technology has made it easier than ever both to engineer...is true when designing gRNAs for using CRISPR technology – the “best” gRNA depends an awful lot on what...Gene knockout by NHEJ Gene knockout with CRISPR technology is usually accomplished by Cas9-mediated dsDNA... Created with BioRender.com. Two newer technologies offer an alternative to HDR for introducing ... another target C or A in the window. Another technology, prime editing (reviewed in Anzalone et al., ... although these are still early days for this technology, and the user may need to optimize numerous parameters...CRISPRa (activation) and CRISPRi (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near...

  10. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ... permeabilization techniques — and so it may be necessary to try multiple techniques. For example, we... ability to detect endogenous levels of the target protein. It may only be able to detect the target protein... (or refute) that the antibody is selectively detecting the target-of-interest in your assay and sample-of-interest...must first confirm that a candidate antibody can detect the antigen of interest in the given protocol. ...supportive, the data must show that the antigen being detected in the intended application — here, flow cytometry...conclusively prove the ability of an antibody to detect the antigen of interest when expressed at endogenous... approach, you’re looking for an antibody that detects the target in the parental cell line, but not in...

  11. Common Injection Routes in Mice

    Type
    Blog Post
    ...the mice, these “no needles” techniques may come in handy. These techniques are referred to as “instillation...to conducting mouse experiments. Some of these techniques are what we call “beginner”, or injections that... practice after initial mouse training. Other techniques require more skill and additional training, going...Image created with biorender.com.   Needle techniques Intravenous Intravenous (IV) injections are most...easier of the two, but it actually requires more technical skill to place the needle in the correct vein ...are probably the most advanced needle injection technique. Intrathecal injections are used when the substance... ml (10 µl) Skill required: expert No-needle techniques The term “injection” is most often associated...

  12. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    ...Importantly, conventional flow cytometry uses one detector to detect one fluorophore and therefore one cellular...fluorophores can “spill over” into detectors that are not the primary detector for that fluorophore. For example...single (primary) detector to identify a fluorophore, spectral flow cytometers use all detectors for all fluorophores...panels: five laser spectral flow machines have 64 detectors, allowing for the use of up to 64 markers in a...emitted by a fluorophore is guided to a single detector using a series of mirrors and filters. Importantly... FITC signal is spilling over into the primary detector for PE. In order to correct for this, compensation...the fluorescent molecules can be quite broad, detectors will usually only pick up a small portion of that...

  13. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    ... and can be used in a multitude of scientific techniques such as molecular cloning or molecular diagnostics...used for an array of different molecular biology techniques and experiments. End point PCR’s goal is to amplify... cloning to create a plasmid of interest, for detecting the presence of an insert or piece of DNA in a...reaction (qPCR) also known as real time PCR is a PCR technique used for measuring a starting DNA concentration...during the PCR reaction cycles is continuously detected via a fluorescent signal. The signal increases...concentration. The cycle in which the unknown DNA is detected compared to the standard curve can be used to ...right. ddPCR uses a water-oil emulsion droplet technology that fractions the PCR reaction sample into approximately...

  14. Getting to Know Expansion Microscopy

    Type
    Blog Post
    .... However, most SRM techniques also require specialized instruments and technical expertise, limiting ... model systems, paired with SRM techniques, and adapted for detecting nucleic acids as well as proteins...biological features previously only visible via techniques like super-resolution microscopy or electron ...Super-resolution microscopy (SRM), a family of imaging techniques developed to break through the resolution barrier...some of the limitations?  A “super-resolution” technique that uses common reagents, straightforward protocols...there is always a catch - some limitation of the technique that users should be aware of - and ExM is no ...microtubules. This issue is shared by other SRM techniques and can be alleviated by maximizing label density...

  15. How to Be an Excellent Trainee

    Type
    Blog Post
    ...learning a new technique while visiting a lab, or maybe even training on a new technique with a coworker...research group, you will also typically receive technical mentorship from a ‘bench mentor’. In some instances...mentor is a staff scientist, postdoc, lab manager, technician, or a graduate student. This is the person you...basis who will help you get on your feet with techniques in the lab (and possibly much more, depending...difficult for you to become proficient in lab techniques, which will hinder your progress. You will also...serve two purposes – familiarize you with the techniques you will be learning and the biology you will...there for you to be independent performing the technique you trained to do? Walk through the protocol mentally...

  16. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    ...Förster) Resonance Energy Transfer, technique which allows the detection of molecules' interactions, modifications...fluorophores to proteins, pioneers of this technique were able to directly detect protein/protein interactions in...dissociations in situ. Used since the mid-90s, this technique has revolutionised the way we apprehend molecular...playing “live.” Indeed, FRET was one of the first techniques which enabled the measurement of single molecule...microscope. Historically, molecular interactions were detected by indirect means often using probes with the ... fluorophore can be measured using microscopy techniques. FRET measurement sensitivity makes it suitable...DNA and proteins. FRET, in theory, is an easy technique, but it is very important to follow some simple...

  17. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...to get right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself...Most people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant...you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you...transfer. Troubleshooting The two most common (non-technical) issues with transferring are smaller proteins...than just high background, your issue is likely technical. White circles, for example, could indicate that...incubation steps.  Many errors can arise from technical issues. The good news is that such errors are ...means the amount of protein loaded is outside the detectable range of that antibody dilution. Adjust the antibody...

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

    Type
    Blog Post
    ...chromosome, which was technically challenging. Other homology based technologies We've described Sequence...alternatives to restriction enzyme cloning. These newer technologies have become more and more common, and for good...Overview of Gibson assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues... 5 days. One drawback to the Gibson assembly technique is that the process works best with fragments ...Gibson assembly combined with the popular CRISPR technology and is described in a recent publication by the...When followed by the standard Gibson assembly techniques described above, this resulted in the direct ...Cloning (System Biosciences) In-fusion Cloning (Clontech) Although these kits may come at a high price...

  19. Luminescent Imaging with Nano-lanterns

    Type
    Blog Post
    ...Fluorescent imaging techniques have become indispensable tools for molecular and cell biologists over... Osaka University have developed Nano-lantern technology. Nano-lanterns contain a Renilla luciferase variant...but it has precluded widespread adoption of the technology. In 2012, Saito et al. described the first Nano-lantern...luciferase (RLuc8) with Venus. This luminescent technology is based on the principle of bioluminescence ...to excite the fused fluorescent protein. This technique is very similar to the widely used FRET, but it...ATP sensors based on Nano-lantern, showing the technique’s versatility and adaptability to many different...optogenetic system. Biosensors based on Nano-lantern technology are also very robust, with an eNL-based calcium...

  20. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    ...please read on to learn more about this exciting technology from the folks at Transposagen. This post was...engineering approaches. Most importantly for this technology, however, is that the transposase also enables... with no known upper limit [1]. The piggyBac™ technology can be used for numerous applications, including...of interest. Fortunately, by combing nuclease technology with the piggyBac™ system, these challenges can... DNA using the piggyBac™ transposon selection technology using a site-specific nuclease to stimulate homologous...of using piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette. While...summary, the piggyBac™ transposon is a powerful technology that may be used for a variety of research applications...
Showing: 141 - 160 of 795 results