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We narrowed to 1,009 results for: PLE

Showing: 241 - 260 of 1009 results

  1. 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...

  2. Plasmids 101: Blue-white Screening

    Type
    Blog Post
    ...are a few examples. Make sure you use a proper plasmid (i.e. contains the α-complementation cloning MCS...isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds and inactivates the lac ...to a lacZΔM15-mutant bacterial cell in trans complements the mutation allowing for a functional enzyme...enzyme. This process is called α-complementation. The system described above was put to practical use in the... the following way. Scientists engineered a multiple cloning site (MCS) into the α-peptide (represented... inserted it into a plasmid, creating an α-complementation cloning vector. When a cloning reaction goes...interrupted as shown in cell A, and thus will not complement the cell's β-galactosidase mutation. An unsuccessful...

  3. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    ...benefits gained by lowering the complexity of the screen. For example, the analysis becomes easier, the...discussed below. The software could also be used as a simple tool to rapidly find one or a few gRNAs suggested... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...-defined, hypothesis driven research question. Examples of custom CRISPR screens Differentially expressed...a lasting impression on me. The slide showed a complex signaling pathway with many receptors, signaling...be very useful for drug discovery, to dissect a complex signaling pathway, and to identify what part of...potential components of a signaling pathway. For example, one could study the cellular response to a cytokine...

  4. Five Popular Model Organisms

    Type
    Blog Post
    ...researchers develop a variety of therapies. For example, many scientists use mice to study diseases such...such as cancer since mice better recapitulate the complex interactions between cancer cells, therapeutic ... in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study how different...Yeast (Saccharomyces cerevisiae) Yeast, one of the simplest eukaryotic organism, is a commonly used model ...in breads and other baked goods! Yeast is cheap, simple and easy to work with as they can survive in a ...diseases that are caused by a specific gene. For example, several genes mutated in human cancers have a ...recapitulate in vivo conditions similar to humans. For example, due to their simplicity, yeast have become an ...

  5. RNA Extraction Without A Kit

    Type
    Blog Post
    ...meant for isolating RNA from rat spleens in which they homogenized spleens in a guanidinium thiocyanate solution...tissues, cells, plants, fungi, or bacteria, keep samples cold and work quickly to mitigate RNA degradation...180°C for at least 4 hours.   If your final RNA sample(s) are resuspended in water or TE buffer, store...freezer.   RNA extraction methods evolved into a simple protocol still used today There are many alternative...for RNA extraction and purification. There is one simple method that works, and variations to that method...requires a lot of time and depending on how many samples you may have, access to one or more large, expensive...effectively separated from DNA and proteins by a simple extraction protocol with guanidinium thiocyanate-phenol-chloroform...

  6. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ...nucleotides (for example, Digoxigenin-labelled Uracil) that will bind to complementary mRNA within an embryo...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...specific antibodies that detect the mRNA-riboprobe complex are added to the tissue. The last step involves...cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue. Students...organism of interest, use the RNA to create a complementary DNA (cDNA), and then use PCR and molecular cloning...cell types or entire organs in planarians. For example, the gene Piwi labels neoblast cells and the gene...one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA to bind...

  7. Transferable Skills: Negotiation

    Type
    Blog Post
    ...negotiation as something done not by people wearing a lab coat, but instead by people wearing business suits discussing...though three examples depend on many specific details about the individuals, each example provides a scenario...scenario where both people get something valuable. Importantly, as the third example shows, the ultimate... is important to people so that you can approach negotiations creatively with people from anywhere in ...transferable skill? Negotiation as a zero sum game Some people think about negotiation as a zero sum game - that...machine? Let’s walk through a more specific example. Perhaps you and your lab mate are negotiating ...your organization. In my non-academic roles, some examples of negotiation have been: A job offer conflicts...

  8. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    ...inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-modified from...probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must..., the widespread success of CRISPR-Cas9 across multiple model organisms (3) lies in the versatility of...inject cells with in vitro preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids...which could be less desirable if one has to target multiple sites in a single experiment. Also, the NGG PAM... an optimal distance from the target site. For example, NGG may not be present in AT-rich regions of the..., I was going through NgAgo google forums where people write their experiences with the new method and...

  9. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    ..., and conservation. But please be aware that there are many easy-to-implement confinement strategies that...with wild ones. For example, Drosophila lines with compound autosomes are completely infertile when mated...organisms with Cas9 risk creating a gene drive? The simplest way to make a gene drive is to insert an endonuclease... cell can create an RNA-guided gene drive. In principle, this event could happen even if there isn't any...authorities) was to detail these robust and easy-to-implement confinement strategies2. Our hope was that anyone...laboratory organisms but not wild organisms. For example, an sgRNA-only drive will spread exclusively through...with that sequence2. Both methods are easy to implement and have been tested in yeast5. Ecological confinement...

  10. SciComm: Taking Science to Elementary and Middle Schools

    Type
    Blog Post
    ...I was careful to keep it relatively simple. I honed the complexity of the talk by running it by the teachers... Association (PTA) has been instrumental in supplementing that curriculum. It’s through the PTA that I...elementary students can ask. It doesn’t just need to be people who think of themselves as “scientists.” We had...for my school’s Math & Science Night. There are plenty of places to get ideas for science activities. ...Spangler Science The Fab Lab Middle school: supplementing the science curriculum Sharing my expertise ...will likely change for next time is giving fewer examples and think of a way to make this section more interactive...did not try to sugarcoat the potential dangers of people using the technology in an unethical manner, but...

  11. Antibodies 101: Epitope Availability

    Type
    Blog Post
    ...target protein? Epitope Availability The answer is simple: epitope availability. An antibody can only bind...the researcher and ones that happen naturally.  Sample Denaturing  First, let’s think about induced conformation...frequently denature the proteins as part of the sample prep. This involves using heat and/or chemicals...being used as a marker for flow cytometry, for example, can only be recognized by antibodies that bind...epitopes available if it is being selected out of a complex mix of proteins that have been extracted from a...Picking the Right Antibody  It can get even more complex if you’re looking for proteins that are interacting...help to visualize an abstract protein during your sample prep process, imagining how it starts and if any...

  12. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

    Type
    Blog Post
    ... strategy is that large complexes may not permeate through your tissue samples as homogeneously as a single...processing and sample preparation can also help reduce the levels of endogenous IgGs in your samples, but know...option is from the same species as your tissue samples. The bad news: this situation is hard to avoid ...The culprits are endogenous IgGs in your tissue sample. Most common IHC protocols use two antibodies -...targets.  Pre-formed primary and secondary antibody complexes (Figure 2B) Some labs have found that you can ...with serum from the same species as your primary/sample, and then applying this mixture to your tissue ...secondary antibodies before adding them to your sample so the secondary antibodies aren’t free to bind...

  13. Plasmid Cloning by PCR

    Type
    Blog Post
    ...In its simplest form, PCR based cloning is about making a copy of a piece of DNA and at the same time...few quirks specific to the PCR cloning process - please check out that post if you need a more detailed...detailed refresher on the downstream steps. For this example, we will describe how to copy a cDNA from one vector...location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere ... buffer, as this will save time later In our example, we will use EcoRI and NotI to ligate our cDNA ...start codon (ATG) to the stop codon (TGA, in this example). Assuming you are amplifying from plasmid DNA ...design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...

  14. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
    ...you only need 1-2 ul of sample and the run time is just a few minutes per sample. During the run, DNA fragments...absorbance. Then you measure the absorbance of the DNA sample.  These absorbance measures give you an idea of...dilution factor x 50 ug/ml This method is quick and simple and doesn’t require any special reagents. However...especially when you expect low concentrations in your samples and are often used to quantify DNA for next generation...fluorescence-based methods require a standard curve, a set of samples with a known DNA quantity and their corresponding...That way, you can compare the fluorescence of your sample against this curve to quantify your DNA prep. Though...yourself as many fluorometers will calculate your sample concentration for you. Agarose gel electrophoresis...

  15. Hot Plasmids - February 2022

    Type
    Blog Post
    ... amino acids, HA is nine), multiple repeats of the tag can recruit multiple frankenbodies to amplify the...ever like to write about a recent plasmid deposit please sign up here.  Here's what you'll find in this...genetic screening technique using Cas12a for multiplexed gene perturbation The Penn State Protein Ladder...Anti-FLAG frankenbody (FB) construction from the complementarity determining region of an anti-FLAG scFv and...genetic screening technique using Cas12a for multiplexed gene perturbation by: Gabrielle Clouse While ...method for screens. Cas12a’s ability to perform multiplex editing within a single expression cassette is...vs. Cas 12a expression cassettes when encoding multiple guides. While two guides require two arrays for...

  16. Evolution of Lab Techniques

    Type
    Blog Post
    ...fluorescently labeled probe. If the DNA is complementary to the probe (for example, has the sequence of the mutated...searching for), then the probe will bind to the sample thereby labeling it. The label can then be visualized...strength of the signal in comparison to a control sample. Southern blotting applications range from confirming...experiments to forensics and clinical diagnostics. One example of southern blotting being used to identify a gene... MstII and southern blotting are conducted on a sample from an individual without sickle cell anemia, ...DNA sequences to see which is more abundant in a sample. Whereas Southern blotting is labor intensive and...different restriction enzyme digests. Incubating a DNA sample of unknown sequence with restriction enzymes and...

  17. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ...mutations. Multiple gRNAs could be used to combat this problem, just as ART includes multiple drugs to ...Over 25 million people worldwide are currently infected with the lentivirus HIV-1. Today, HIV-1 can be...undetectable in the blood. But the virus doesn’t completely disappear; it just hides in latently infected...accomplish this tough job! Kamel Khalili’s lab at Temple University has demonstrated two potential strategies...the immune response. The second strategy is the simple removal of the HIV-1 genome from infected cells...transcriptional activation. Zhang et al. designed multiple gRNAs to target the 5’ long terminal repeat (LTR...activation of HIV promoter-luciferase constructs. In multiple HIV-1 latent T cell lines, as well as a latent...

  18. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    ... as these immune complexes are smaller than a primary:secondaries:conjugates complex. If you are trying...Antibody-based assays are complex and antibodies may perform differently in different samples and experimental ...primary (linear) structure using heat or acid, for example in a Western blot. If you were to try and use such...all IHC experiments are the same. Differences in sample processing, fixation, antigen-retrieval, and permeabilization...chance that the antibody could meet your needs.    Multiplex experiments Finally, consider whether you will... in the experiment. If you are planning to use multiple antibodies in a single experiment then you will...antibodies are sold under different names through multiple vendors, so look at epitope target, source, and...

  19. Tagging Optogenetics and Chemogenetics Receptors: Fluorescent Proteins and Other Options

    Type
    Blog Post
    ...reveal the hM4Di coupled to mCherry (top) or HA tag (bottom). The images show examples of dendrites (blue...interest. Furthermore, fluorophore variants (for example green and red fluorescent proteins) allow simultaneous...properties or trafficking of the fusion protein (Kimple et al., 2013). We found that the fluorescent proteins...The hM4Di receptor is an engineered Gi protein-coupled receptor, one of the Designer Receptor Exclusively...antibodies against mCherry, and secondary antibodies coupled to nanogold particles, that allow a high-spatial...needed, the use of HA and other short epitope tags (Kimple et al., 2013) could help improve the transport ... not generalizable to all fusion proteins. For example, we saw that another type of DREADD, hM3Dq, is ...

  20. Antibodies 101: Fab Fragments

    Type
    Blog Post
    ... antibody complexes before applying them to samples, such as when performing IHC in a sample where your...gives you a better chance of getting the complex into the sample. Alternatively, Fab fragments can be used...proteolytic approaches. This comes in handy when, for example, you want to swap out one isotype for another. ...antibody in isolation from another portion. For example, Fab fragments are molecules composed of just the...antibodies. Their smaller size allows them to get into samples more efficiently and can also improve localization...antibodies bound together may be too large. Using a complex that is one full antibody bound to a Fab fragment...same species as your secondary antibody to your sample prior to adding your primary and secondary antibodies...
Showing: 241 - 260 of 1009 results