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We narrowed to 876 results for: NSI;

Showing: 121 - 140 of 876 results

  1. Lentiviral Vector Guide

    Type
    Guide
    ...plasmids (mostly third-generation) can be used in transient transfections, but expression will be greatly ...plasmids cannot be used in direct transfections for transient expression of a transgene. For more detailed protocols...stable expression and wide tropism. Addgene has an extensive collection of lentiviral CRISPR libraries, including...initial infection. Third-generation systems are considered safer than second-generation systems because ...transfer plasmid is an oncogene. Additional consideration for oncogenes should be made on a case-by-case...common points of entry. Biosafety should always be considered with respect to the precise nature of experiments...9765382 Durand, S., & Cimarelli, A. (2011). The inside out of lentiviral vectors. Viruses , 3 (2), 132...

  2. Adenovirus Guide

    Type
    Guide
    ...a protein capsid and the viral genetic material inside. Adenoviral genomes range between 26–45 kb in length...ITRs is limited by the physical space available inside of the adenoviral capsid and directly correlates...used generation for research purposes. The system consists of two plasmids that eventually recombine to form...expression delivered through rAdV vectors is normally transient and short-lived. Second- and third-generation ...common points of entry. Biosafety should always be considered with respect to the precise nature of experiments...the Adenoviridae family. Episome Genetic element inside the cell that remains as a part of the eukaryotic...

  3. Promoters

    Type
    Guide
    ...equivalent called the Pribnow box which usually consists of the six nucleotides, TATAAT. Proximal Promoter...position, simply titled the -35 element, typically consists of the sequence TTGACA and this element controls...have also been discovered in eukaryotes. Operons consist of a promoter, which is recognized by the RNA polymerase...transcribes messenger RNA (mRNA) which is the RNA responsible for providing a stable template for the translation... RNAs (tRNA), the adaptor molecules that are responsible for bringing amino acids to the ribosome when...

  4. Molecular Biology Reference

    Type
    Guide
    ...additional strain-related information and a more extensive strain list. Strain Vendor(s) Genotype BL21 Invitrogen...sequencing chromatogram which provides the color and intensity of each fluorescent signal. Sanger can sequence...and C always pairs with G. RNA on the other hand consists of Adenine, Cytosine, Guanine and Uracil (U). ...

  5. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...transfer plasmid is an oncogene. Additional consideration for oncogenes should be made on a case-by-case...common points of entry. Biosafety should always be considered with respect to the precise nature of experiments...

  6. Technical Design of a Western Blot

    Type
    Blog Post
    ...chemicals, and are not always consistent. Precast gels, while expensive, reduce protocol time, can be ...chemical-based blockers, while much more expensive, are consistent and stable at room temperature for 1–2... Direct detection method Less sensitive; usually more expensive Nitrocellulose or low-fluorescence...Multiplex Fluorescent proteins Less sensitive; more expensive Strip/Reprobe PVDF membrane Increased...specificity, lower cross-reactivity, and are more consistent lot-to-lot. The highest reproducibility comes...quantitative assays. On the other hand, they are not as sensitive to lower amounts of protein, and can require more...  Lysis buffer When choosing your lysis buffer, consider the subcellular location of your protein. Proteins...

  7. RANbodies: Reporter Nanobody Fusions

    Type
    Blog Post
    ...antibody is less sensitive than HRP RANbody detection, it is approximately as sensitive as detecting the...detection is less sensitive than HRP RANbody detection but approximately as sensitive as antigen detection...approximately as sensitive as detecting antigen with a standard primary/labeled secondary Less sensitive than HRP-RANbody...approximately as sensitive as detecting antigen with standard primary/labeled secondary   Less sensitive than HRP-RANbody...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to...secondary antibody. Chicken IgY This RANbody reporter consists of two chicken IgY domains. Chicken IgY domains...detected enzymatically with HRP substrate More sensitive than detecting antigen with a primary/ labeled...

  8. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...      Figure 1: A protein gradient. The density of the bands, which increase as protein concentration... buffers and strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing...re seeing protein aggregation, you may want to consider changing your incubation temperature. Some proteins...aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, ...If you need very accurate protein measurements, consider using an unstained protein ladder, which will ...ladder on the lefthand side. Whatever you do, be consistent and document it.   Did it work? Save yourself...reversible staining method of choice is appropriately sensitive. If using a PVDF membrane, you can dry the membrane...

  9. Viral Vectors 101: Voltage Indicators

    Type
    Blog Post
    ...detailed below. Voltage Sensitive Domain (VSD) GEVIs Simply put, VSD-based sensors consist of a VSD fused to...gated ion channels all depend on transmitting and sensing electrical potential. Thankfully, there is a tool... These tools have two main elements: a voltage-sensing component and a fluorescent output. The GEVI family...divided up structurally into 2 main groups: voltage sensitive domains and Rhodopsin containing GEVIs. Chemigenetic...fluorescent reporter and the mechanism of their voltage sensing component, which separates them into two distinct...indicator, which can make the kinetics of voltage sensing to signal output slightly slower than other approaches...approaches. Functionally, the voltage sensitivity of the available VSDs has been successful even at the...

  10. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    ...the loading control: the intensity of each POI band is divided by the intensity of the loading control ...band intensity measurements become extremely susceptible to error, since tiny changes in intensity can ...membrane, which would be directly reported by band intensity. Researchers use later steps in this chain (blue...trust Assumption #1, any change in the normalized intensity of your POI could reflect either a genuine change...relationship between protein abundance and band intensity is an S-shaped curve. Sparse proteins may not ...problems for proteins of very different abundance, consider the following example gel. Figure 5: An... on the plot of protein abundance versus band intensity (right). Created with BioRender.com. On this...

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

    Type
    Blog Post
    ...Specific ​H​igh ​s​ensitivity ​E​nzymatic ​R​eporter un​LOCK​ing) technique Sensitivity: Single molecule...Two-step-SHERLOCK has a sensitivity in the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar...for rapid, ultrasensitive and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies...SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-100 copies of RNA per microliter Time: ~40...two crRNAs that bind to two regions improves sensitivity of the assay. This reaction also contains SSB...one-temperature reaction, and is nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection...detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of RNA per microliter Time: Not mentioned...

  12. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    ...conditions. The use of low intensity light solves each of these problems; inexpensive tools such as LEDs can...precise, consistent, and controllable pulses of light. Complicated patterns of light intensity and wavelength... low-intensity light, so off-target effects should be limited. Phytochromes are light-responsive systems...Tabor have been working on making E. coli light responsive since 2005, when Tabor was working in Voigt's... heading of two-component systems (TCSs). TCSs consist of a histidine kinase that phosphorylates a response...Christopher Voigt’s lab created the first E. coli light-sensitive two-component system (TCS). Levskaya et al. fused...Tabor et al. subsequently engineered a second photosensitive TCS activated by green light; these two systems...

  13. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...published Aug 30, 2018 and updated April 16, 2020. Sensitive and specific nucleic acid detection is crucial...nucleic acid detection however, either lack the sensitivity or the specificity to detect nucleic acids at...at low concentrations and/or are too expensive, time-consuming, and complex to use outside of standard...and Cas12a, to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at...and optimize the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2...CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity Enzymatic Reporter UnLOCKING Cas13 (C2c2) was... your targeted nucleic acid. To increase the sensitivity of the assay, targeted DNA or RNA from a sample...

  14. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ...are not as sensitive, however, so those studying low abundance antigens may want to consider alternative... ELISAs for their accuracy, specificity, and sensitivity (a good ELISA can detect picogram quantities ...advantages and disadvantages of each, and some considerations when using the method. ELISA basics All ELISAs...detectable product. Scientists compare the signal intensity of the sample to that of a standard curve to extrapolate... themselves, the process can be laborious and expensive. In some cases, conjugation methods can interfere...Incorporating a secondary antibody increases the sensitivity of the assay, since multiple secondary antibodies...sandwich ELISAs, in a competitive ELISA the signal intensity is inversely proportional to the amount of antigen...

  15. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    ...retain fluorescent intensity during exposure to light, is another factor to consider. If your application...interiors to many inquisitive scientists. Seeing inside these specimens has offered illumination on biochemical...will outline some fundamental characteristics to consider when selecting a dye or ligand to make your selection...proteins would be SNAP-tag® and HaloTag®. These consist of a genetically encoded enzyme variant tag that...is bound through green light emission. Key considerations in selecting fluorescent dyes/ligands Effects...-conjugation affects protein function and its propensity to bind cellular components. Some dyes target...stably combined with nanoparticles, maintaining intensity at ~85% of its original value after 60 minutes...

  16. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...subcloning, expansion, and confirmation. This procedure can take several months but should be considered in labs...benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here...results in less than a day, but tends to be less expensive than an ELISA kit. In this approach, viral RNA...lentiviruses in parallel, this approach should be considered. Similar to the p24 assay, titering via measurement...When using qPCR based methods for viral titration consider using a reverse transcriptase mutant in a separate...carryover. Functional titer Functional titer is considered a more accurate method to quantify virus as it...of this approach is that it can be quite labor intensive. Target cells are transduced with serial dilutions...

  17. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    ...the least expensive of all the options discussed as it only requires PCR and the inexpensive DpnI enzyme...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...the following cloning methodologies have been extensively described in other articles; so if you are interested...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...

  18. Hot Plasmids: FRET-Based Biosensors

    Type
    Blog Post
    ...cellular-level events and behavior. It’s a problem that he considers “one of the greatest challenges of neuroscience...particular advantage for observing events taking place inside cells that are on the move, such as T lymphocytes... calcium-binding sites as possible for added sensitivity. As reported in Nature Methods, the FRET changes...essentially all of the standard organisms for which extensive genetic tools are available. While he recognizes... he said. “It gives you more information than intensity-based readouts.” Griesbeck also points out that...approach he and his colleagues took to optimize sensitivities can now be applied to dozens of other published...for visualizing the transcription factor cAMP-responsive element-binding protein (CREB) in living cells...

  19. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...different opsin (natural and engineered proteins responsive to light in combination with retinal) variations...reviewed in Abreu & Levitz, 2020), including light-sensitive chimeric GPCRS (OptoXRs) (Tichy et al., 2019),... the extracellular domain of endogenous light-sensitive receptors, like mammalian rhodopsin, and the intracellular...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...

  20. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    ... One consists of NLuc fused to enhanced green fluorescent protein (GpNluc) and the other consists of NLuc... (FRET) probes. These probes generally consist of a responsive protein (e.g. calcium binding protein) ...membrane-inserted voltage sensing domain (LOTUS V; Luminescent Optical Tool for Universal Sensing of Voltage). With...how bright you can make a reaction but for how sensitive you can make a reaction. NLuc requires 100-fold...) fused to two fluorescent proteins. When the responsive protein binds to calcium it changes conformation.... In well-designed FRET-based biosensors, the intensity of the FRET signal can be correlated to specific...No photoexcitation High Ion specificity High sensitivity Easy expression Gives ratiometric data The result...
Showing: 121 - 140 of 876 results