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We narrowed to 1,010 results for: PLE

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  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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...

  7. 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...

  8. 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...

  9. 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 ...

  10. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ... is completely degraded. Cloning Grade DNA (high quality/low quantity) – You can usually complete a cloning...transfection-grade While we’ve included a sample of some commonly used kits, please note that Table 3 is certainly...plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if you get a ...easier than others to prevent quality issues. For example, some kits reduce the risk of ethanol carry-over...you mix specially coated magnetic beads into your sample and use magnets to isolate the beads for the wash...Take your average column-based miniprep kit for example. These kits are designed to handle ~1–5 mL of liquid...can affect the lysis buffer ratio and result in incomplete lysis. You may be able to scale the amount of...

  11. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...dish can be easily applied to multiple dishes, remember that handling multiple dishes will take longer and...packaging systems divide the viral elements among multiple plasmids. Second generation packaging systems ...transfer gene. By dividing the viral elements across multiple plasmids, the risk of recombination and consequently...different generations of lentiviral packaging systems please see Addgene’s Lentiviral Guide. While the third...plasmid are not always the best for others. For example, in our hands we find that a double harvest approach...larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios can affect ... volume than with the unconcentrated prep. For example, at Addgene we typically concentrate 100-fold meaning...

  12. Split-BioID: An Improved Method for Studying Protein-Protein Interactions

    Type
    Blog Post
    ...biomolecules. They can be part of multiple dynamic and overlapping complexes that have distinct functions....biotin-containing media. FRET is another example of a protein fragmentation complementation assay that can be used to...proteins that interact with a particular protein  complex as opposed to an individual protein. The Bethune... when the bait is part of a particular protein complex. Not performed in a native cell context. For AP-MS...experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method, each...Protein Interactions. Split-BioID is a protein complementation assay where BirA*, an E. coli biotin ligase...proteins Y and Z interact, the two BirA* fragments complement each other to form a functional BirA* enzyme....

  13. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    ...membrane will remove some of your sample —and harsher stripping means more sample loss. If you are still optimizing...you want to detect more than one protein in your sample? Or what if something weird happened during your..., almost like new. This approach preserves your sample and saves you from having to run a whole new gel... of time that you want to probe your blot with multiple antibodies. You’ll want to set up your blot with...stripping solution (see below) to minimize loss of your sample from the membrane. Next, plan out the order of ...stripping methods. If you are planning to detect multiple proteins through several rounds of stripping and... the weaker binding ones first to preserve your sample. Pro tip! Because some protein is lost each time...

  14. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...plasmid deposit process can be broken down into four simple steps: Send plasmid information to Addgene Material... explain how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — ... “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with Addgene... a collection that is greater than 75 plasmids, please contact us at [email protected]. These deposits...any functional consequences of that change. For example, “Mutated aspartate 123 to Lysine (D123K), lowers...indicate type in “Vector Type Other” column. For example, you may enter “donor template vector”. Cloning...37 °C, or room temperature. Growth Strain Please indicate which growth strain Addgene should propagate...

  15. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ...with increasingly complex experimental conditions. The ability to construct more complex, multi-fragment...fragments in a single “one-pot” reaction to form complex, multi-insert modular assemblies that enable biosynthetic...enzyme cutting results in 5’ four base overhangs complementary to the overhangs of the assembled inserts. After..., the BsaI site is no longer present, allowing multiple fragments to be assembled in an ordered, pre-determined...assembly Intermediate 5- or 12-fragment assembly More complex 24-fragment assembly Single insert cloning efficiencies...Sequencing of blue colonies showed the expected complete sequence for the lacI/lacZ genes (1), while sequencing...single omission should block the formation of a complete assembly that would result in a blue phenotype...

  16. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ... DNA and processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed...questions are especially important when producing multiple vectors in parallel because the risk of accidental...in-house sequencing capabilities and Seqwell’s plexWell technology, we can now perform NGS at Addgene ...these known impurities, as was the case for clean sample AAV-44362. In addition to these expected hits, ...to “random” genes. Do these hits always mean the sample is contaminated? No - what really matters is the...of hits to the same sequence (gene). In a clean sample, there are typically fewer than 10 hits to a given...>100 hits - immediately raises a red flag. For example, AAV-68544 contained 220 hits to the CHRM4 gene...

  17. Experimenting with New Careers while in Grad School

    Type
    Blog Post
    ...academia One recent example: Don't call me a dropout: Why science needs more people to quit the lab. ...re provided throughout your PhD program.   Find people who will support your professional development ...are not as supportive of your career goals, find people who are. I wanted to do fun science activities ...activities with the public, so I reached out to people who were educating the public and organizing events. I contacted...attention on social media. I gained a concrete example to use to promote future attempts and learned a...try new things, develop new skills, and meet new people. There were visiting seminar speakers each week...meet with various professionals, and there were multiple avenues for professional development including...

  18. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

    Type
    Blog Post
    ... useful they are implemented in most materials that have been engineered. For example, Boole’s logic shows...biological engineering openly so as to benefit all people and the planet. For his many efforts in open science...BioBricks Foundation to distribute the physical DNA samples, for when that's the preferred mode of receiving...phrase we like to use is 'the parts must flow.' People must be able to use genetically encoded functions... for engineering an entire cell. Typically when people use plasmids, they are trying to do one thing at...Integrase Logic (BIL) gates collection of parts implements the basic logical functions, which we know and..., computers, and now through BIL gates we've implemented them in DNA. We thought these were so much fun...

  19. Cre-ating New Methods for Site-specific Recombination in Drosophila

    Type
    Blog Post
    ...to make complex genome modifications in Drosophila using multiple recombinases. To make multiple, precise...recombination by KD, B2 and B3 resulted in complete or near-complete excision of the stop sequence. In contrast...to DNA damage and developmental aberrations. In multiple systems, Cre itself, without the presence of a...toxicity, but with the caveat of partial rather than complete recombination. If you’re looking to use site-specific... high recombinase levels. These data show that multiple recombinases can be used together to designate...for specific recombination patterns that require multiple recombinases. These tools should further enhance...editing capabilities in Drosophila. References: Multiple new site-specific recombinases for use in manipulating...

  20. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    ... (5,12,13). For example, in the Ligase Detection Reaction (LDR), probes complementary to the sequence ... extended to multiplexed probe sets that allow the simultaneous interrogation of multiple potential SNP...ligation of fully base-paired nicks with no gaps. For example, DNA assembly methods, such as Gibson Assembly®...without the exo/polymerase generation of annealed complementary regions. Why use a high fidelity ligase? Numerous...extends earlier studies through a high-sensitivity multiplexed format (21, 27). This methodology has been used...separate pools were prepared, each with a different complement strand covering all 16 possible NN pairs in the...Stefan V., and Göran Magnusson. "Sealing of gaps in duplex DNA by T4 DNA ligase." Nucleic acids research 10.5...

  21. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...for multiplexed targeting with Zhang lab plasmids? A6: For multiplexing CRISPR to target multiple genome...4 of the Cong et al. (2013) Science paper. For example, if you want to use double nickase, you could express...want to use wildtype cas9 or double nickase). For example, if you want to target two genomic loci with wildtype... vector, for one well in 24-well plate. A good example/reference is Ran et al. (2013). In the case where...putting in the full target site in the donor. For example, choosing target sites that span the knock-in gene...selection marker to select transfected cells (for example, plasmid with a puromycin resistance cassette, ...individual genotyping (using Sanger sequencing, for example) on each colony in order to screen for positive...

  22. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ... encode which amino acids (see example to the right). With such simple rules, you might think it’s easy...codons unaccounted for. The result is redundancy; multiple codons encode single amino acids. Evolutionary...varied codon preferences among organisms aren’t completely understood, but some possible reasons include...resources for the entire translation process. For example, during high growth rate conditions, E. coli preferentially...encoded by highly abundant, charged tRNAs. For example Tuller et al. found that translation efficiency...translated regions a chance to fold properly. For example Pechmann and Frydman found that tracts of non-optimal...producing and charging specific tRNA pools. For example, tRNAs used in genes encoding amino acid biosynthetic...

  23. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    ...fully summarized in a short blog post, but some examples I’m personally interested in include: intracranial... is an approximation of reward or the sense of pleasure that the animal perceives through taking a drug... also models reinforcement but is considerably simpler than ICSS. The test chamber is divided into two...expression of the early response gene c-fos. For example, using Chr2 stimulation should result in robust...c-fos expression to get the evidence you need. For example, as shown in this study [3], hippocampal neurons...mouse is placed in a novel context. As a proof-of-principle test, the authors were able to show that HR blocked...involved in regulating the behavioral output. For example, if you are interested in studying a brain region...

  24. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    ...will not affect single-plexed experiments, it needs to be addressed when multiplexing. It is imperative that...cannot be indirectly probed simultaneously on the sample. What to do?  If you’ve ever found yourself facing...the constant regions from an alternative isotype (purple, mouse). Depending on the cloning process, all ...conversion, gives users the flexibility needed for multiplexing. In the case above, you can’t costain with your...visualize both targets in parallel (Fig. 2). For example, if both of your antibodies are rat IgG1, then ...antibodies with the same isotype can not be used for multiplexed indirect staining methods since the secondary...the new Fc region. In addition to increasing multiplexing capabilities this process is frequently used...

  25. The Strength of Story Telling

    Type
    Blog Post
    ...on living as a scientist in Beijing, talking to people about taking beautiful photographs of their studies...Story Telling at work Don’t believe me? Here’s an example of storytelling to answer an informational interview...it worked out for all of us.” This is a classic example of the STAR (Situation, Task, Action, Result) interview...than I’m able to produce. But it’s also a great example of storytelling – you get context, time, cause,... and wide, keep your website up to date, invite people to ask questions, be approachable. Engage with ...technique you have in your arsenal to make sure people pay attention. Highlight your lowest points and...into a scientific story, but it will ensure that people pay attention. Practice Nobody gets good overnight...

  26. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    .... HDR is crucial for certain applications, for example, correcting the allele that causes sickle cell ...decision. Our conclusions reinforce a key scientific principle: that understanding a biological process in detail...manipulate that process. We began our research with a simple question: how do Cas9 and sgRNA interact with and...tightly to three of the four strands of the cleaved duplex, while the fourth strand (the released strand in... in Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9...Cas9 binding to the cleaved DNA duplex. Image from Richardson et al., 2016. We next wondered if we ...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...

  27. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    ...that need people who are data oriented, scientifically minded, and able to communicate complex information...research and potential solutions During the first couple of sessions of the symposium it was clear that,...postdocs. Unfortunately, even when universities have simple postdoc numbers sorted out there can be gaps in...Postdoc: An Institutional Perspective. This lack of complete information does not, however, prevent us from...these positions are available and that many of the people who work in them are incredibly happy with their...need to be able to network and interact more with people who have taken a variety of career paths and should...collaborators, others you know in the field, and even people who aren’t in the field. One PI on the panel even...

  28. Behind the scenes: Addgene’s new search engine and more

    Type
    Blog Post
    ...remedy this, throughout the year we rolled out multiple changes to our website, including the recent homepage... new website search. Tackling findability from multiple angles    We assembled a cross-team team, with...interface.   The team approached the issue from multiple angles: the overall user experience of our website...website navigation paths, the organization and completeness of our information, and the accuracy and relevance...relevant to a specific query and category. For example, a search scoped to plasmids shows filters for ...appear in our data into a single filter value. For example, there is only one viral prep filter for promoter..., human synapsin, hSynap. Matching We capture multiple variations of the same information in our search...

  29. dTAG - You're it!

    Type
    Blog Post
    ...degradation with dTAG-13. Example experiments See our recent papers noted below for examples of dose-responses... pharmacological inhibitors including rapidly depleting a protein-of-interest, increasing target selectivity...FKBP12F36V-tagged proteins. The dTAG molecule induces ternary complex formation between the FKBP12F36V-fusion and the...happy to share aliquots of dTAG-13 in the meantime, please address your requests for dTAG molecules and any...parental cells (or cells expressing a control -- for example, an FKBP12F36V-tagged LACZ) with dTAG molecules...5 in Nabet et al., Nature Chemical Biology for examples). Monitor neo-substrates Small molecule degraders...neo-substrates varies in different contexts (for example, IZKF1 and IZKF3 are cell-type specific transcription...

  30. CRISPR Meets Synthetic Biology: A Conversation with MIT’s Christopher Voigt

    Type
    Blog Post
    ...resources of a cell. The objective is to give people the complete toolbox for taking on very large genetic ... do as genetic circuits become larger and more complex? Voigt: If you look at all the products of biology... on genes at different times. It requires more complex synthetic networks. Addgene: What hurdles did you...overcome and what hurdles remain? Voigt: There are a couple of hurdles we haven’t yet overcome. For this particular...CRISPR chemistry for a NOR gate. But there are a couple of problems. You couldn’t do it without repeating...first one worked in each case. Sometimes in CRISPR people talk about off-target interactions. That’s relevant.... There haven’t been enough genetic parts for complexity. We have characterized almost 600 terminators...

  31. Popular Retroviral Vectors and Their Uses in Scientific Research

    Type
    Blog Post
    ...Plasmid Kit Multiple Lentiviral Expression System Kit (Kit #1000000060) The MuLE (Multiple Lentiviral ...several different viral vectors to choose from, for example gamma-retrovirus, lentivirus, Adenovirus, and Adeno-associated...and lentiviral envelope plasmids are listed here. Please note that both lentiviruses and gamma-retroviruses...combined with high-throughput sequencing to perform multiplexed analysis of cells pre-treated with a compound...Transfer Vectors    Expressing your gene of interest pLenti-puro (Plasmid #39481) You can clone your gene ...We are constantly improving our CRISPR reagents. Please check www.genome-engineering.org for the most up-to-date...system facilitates the simultaneous introduction of multiple genetic alterations into mammalian cells. The ...

  32. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

    Type
    Blog Post
    ...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...assembly of the crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the...protein while others can inhibit CRISPR enzymes from multiple species of bacteria.  A note on anti-CRISPR nomenclature... of discovery (Bondy-Denomy et al., 2018). For example, AcrIIA4 mentioned above indicates that it’s the...activity one of three ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM...site (Harrington et al., 2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes ...and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II to the photo-sensitive LOV2 domain...

  33. Gaining Leadership Skills Volunteering at a Professional Organization

    Type
    Blog Post
    ...predecessors. Changes were implemented and I had to adapt accordingly. For example, the quarterly and annual...annual NPA meeting to learn more. Having met many people at that conference, I later became involved with...volunteered for various tasks as things came up. For example, I updated some sections of the NPA’s website. ...volunteering, you can gain experience working with other people mostly online and on monthly phone calls. This ...distance communication and learning to work with people despite the distance improved my verbal and written...understand how to work with many different types of people both above and below you. During my time at the...NPA. You must accommodate many different types of people in order to harness their strengths and work effectively...

  34. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    ...are often thought of as simple organisms , they have many levels of complexity, especially when trying...about it, the more I realized that it cuts across multiple disciplines, which really epitomizes what the ...learning how to grow and care for the worms. As an example of one of the problems we work collaboratively ...We attempt to deconstruct and understand this complexity using existing C. elegans mutants. For instance...data is freely available, and the collection of samples and sequencing is being organized and funded by...allows the project to be open to a wider group of people. This is a perfect starter project for our computer...Kittybiome project gives them access to rich and complex microbiome data for analysis. And, well, cats.....

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

    Type
    Blog Post
    ...things started. Give a couple of audience members a chance to participate. For example, at a recent event ... Reddit AMA Perhaps we should start with some examples of a bad panel discussion? You know, the one where...interesting stories that will make for a fun discussion. People are much better at remembering these things on ... is tough for women in skirts, especially short people – which I am, so I hate stools). I like it when... with an unexpectedly great turnout). This gets people to sit close to the front so the interactions are...interesting discussion. The audience knows these people and bios were likely part of the publicity. Good...science or expertise by listing some and asking people to raise hands in response. I am always ready with...

  36. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...news is there’s a few simple ways to deal with this issue. One way is to grow multiple bacterial cultures...helps me figure out if I have enough plasmid to complete my experiment. AAV is usually the limiting reagent...concentrate the purified virus. This process can be completed in one long day, or the virus can be stored at...aliquots to use for qPCR titering. This helps avoid multiple freeze/thaws of your larger aliquots of virus....particles that are present in an AAV prep. AAV samples are first DNaseI digested to remove any residual...technology or a TaqMan primer/probe set can be used and samples are quantified by comparing to an AAV standard ... plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR...

  37. Plasmids 101: Multicistronic Vectors

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    Blog Post
    ...co-expression of multiple unique genes, you can start with a plasmid that has multiple cloning sites flanking...Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists...co-transfection of two or more plasmids, the use of multiple or bidirectional promoters, or the creation of... are useful almost anytime you want to express multiple genes in the same cell. Although it is possible...to drive co-expression by using a plasmid with multiple, individual expression cassettes, having the genes...viruses to allow for the efficient translation of multiple genes from a single transcript. The two strategies...20 amino acids) and produce equimolar levels of mulitple genes from the same mRNA. The term "self-cleaving...

  38. Tag Your Favorite Yeast Genes with Ease

    Type
    Blog Post
    ...in protein complex studies where tagging of multiple protein products is desired, as multiple selection...cerevisiae and S. pombe. The protocol is surprisingly simple, requiring only a PCR product containing the modifying...for easy capture and detection of proteins and complexes, without the artifacts sometimes associated with...Formosa, at the University of Utah, has built a complete collection of yeast tagging modules with each ... Dr. Formosa has deposited six plasmids with a multiple cloning site in place of the epitope tag for creation...ideal for tandem affinity purification of protein complexes. John Pringle and Jürg Bähler have deposited a... S. cerevisiae, with the additional benefit of multiple selection markers for combining modifications ...

  39. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    ... affected by gene activation rather than depletion. Multiple strategies have been developed to recruit... stranded DNA breaks that commonly result in a complete loss of function while nuclease-deactivated Cas9...and assessed the libraries performance via the depletion of a gold standard set of genes (1,580 essential...effective than other popular CRISPR libraries in the depletion of essential genes. In the subsampling analysis...have several limitations. CRISPRko results in a complete loss of function meaning that if an essential ...libraries utilize an engineered Cas9, which contains multiple point mutations in the nuclease domain. These ...domain such as VP16 to dCas9 or the recruitment of multiple copies of VP16 termed the “Sun Tag” method (Tanenbaum...

  40. MXS Chaining

    Type
    Blog Post
    ...many biological fields. In synthetic biology for example, high-throughput cloning can be used to combine...powerful imaging technologies, the combination of multiple fluorescent protein sequences to develop diverse...one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent... variety of other clever applications. In one example, modular constructs generated with MXS-chaining...Tubulin Repeatable, directional cloning   The principle behind MXS-chaining is repeatable, restriction-enzyme...individual plasmids and are each flanked by the same multiple cloning site (MCS). Modules are assembled one ...being directional, meaning that (in the previous example) one can control whether the third module is added...

  41. What's New in CRISPR - March 2020

    Type
    Blog Post
    ...downregulate multiple genes at once. Read the paper in mBio Find the CRISPRi plasmids A multiplexed gRNA piggyBac...Base editing CRISPRi in Caulobacter crescentus Multiplexed gRNA for human pluripotent stem cells CRISPR-... Blue light renders the NLS accessible and the complex enters the nucleus where dCas9-Mxi1 can repress.... In the dark, the NLS is inaccessible and the complex remains in the cytoplasm. Read the paper in Cellular...differentiation, but it takes several weeks to delete or deplete individual genes. Michael Laub’s lab developed ... deliver uniform and sustained expression of multiplexed gRNAs in human pluripotent stem cells. Previously... construct and the fluorescently-labeled gRNA multiplex. Read the paper in Scientific Reports Find the...

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

    Type
    Blog Post
    ...field. Fundamental principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores...attached to self-labeling proteins. Some common examples of these self-labeling proteins would be SNAP-...of protein substrates/peptides and antibodies. Example applications of this include coupling to an antibody...specific receptors is key, especially for G-protein-coupled receptors (GPCRs). Mutations in receptor sites ...photons absorbed over the photons emitted. For example, DAPI is much dimmer than Alexa Fluor 488, and ...signal and facilitate clear differentiation between multiple dyes during analysis. Image resolution is impacted...microscopy techniques, such as Stimulated Emission Depletion (STED), and Stochastic Optical Reconstruction ...

  43. An Inside Look into Addgene's Dev Team

    Type
    Blog Post
    ...approaches that may come up during discussion.  One example of agility at work is our process for working on...instructions on how to demonstrate the feature. For example, we recently reduced the number of steps involved...stories in that sprint relate to the theme. For example, we could have a shipping sprint and all the stories...get different perspectives on how things are implemented and from that, develop better software.  Maintenance...how you combine it with other ingredients.  For example, we use a third party library to handle label printing... compatible with the updated library. A recent example is updating our software from Python 2.7 to Python...analogy. Human languages evolve over time, for example from Ancient Greek to Modern Greek. Books written...

  44. Mesothelioma - Causes, Symptoms, and Treatment

    Type
    Blog Post
    ...there are some genetic factors involved. For example, multiple studies show that certain mutations of the...is a terrible disease that is, unfortunately, completely preventable. Mainly caused by exposure to asbestos...mesothelioma instead rely upon private donations. Many people think that because mesothelioma is so rare, there... researchers don’t entirely understand why some people develop the disease while others never do, but ...slow the growth of tumors. More often than not, multiple techniques are used as part of a treatment plan...positive potential for treating mesothelioma. For example, patients who have peritoneal mesothelioma – that... be used to distinguish between three groups of people:  Those who have mesothelioma Those who have been...

  45. Using Video to Share Your Science: We Share Science

    Type
    Blog Post
    ...fields in order to provide useful solutions to complex challenges. As a cross-disciplinary researcher,...Many of the challenges in science today are too complex for any single discipline to solve them on their...interdisciplinarity index of research for single or multiple disciplines – you can select the disciplines and...few years, I decided to look at what online tools people generally like to use to share ideas – with the... in terms of communicating ideas. In 2012, for example, 3 billion hours of video were watched each month... disciplines leading the way, the future of complementing published research with video overviews was ...networking, visual appeal, instant sharing across multiple other sites), I created a new platform for sharing...

  46. Understanding GPCR Signaling Using TRUPATH

    Type
    Blog Post
    ...stimuli. One way they do so is through G protein-coupled receptors (GPCRs), receptors that play many roles...GPCRs (Takeda S et al., 2002) and many downstream complexes involved, it can be difficult for scientists to...in sight (Olsen RHJ et al., 2020). A transducer complex consists of a trimer of three subunits: alpha, ...collectively known as a heterotrimeric G protein. This complex converts a signal received by a GPCR on the cell...is being able to identify specific transducer complexes that are activated in response to a ligand binding...inactive, these subunits are found in a trimeric complex bound to the intracellular surface of the GPCR....Following activation in response to the ligand, the complex will disassociate, resulting in a decreased GFP2...

  47. Science Careers: Unruly Interests Feed Many Paths

    Type
    Blog Post
    ...career Developmental biology caught me early on: a simple egg is packed with information. I am fascinated...individual diversity to get from gamete to a visibly complex - and unique - adult. My initial research directions...teacher was looking for an outlet. I started asking people: why do you do what you do? How do you like it?...it? how did you get there? Many of those people I met through AWIS (Association for Women in Science); ...visiting seminar speakers. My eye was caught by a couple of unusual job openings, although they seemed too...applied research. I focus in general terms on a couple of very broad topic domains, but each day brings...and surprisingly diverse manuscripts. I work with people, nurturing networks, as much as with technical ...

  48. Replacing Paper: Tips for Choosing an Electronic lab Notebook

    Type
    Blog Post
    ...adopting simple note-taking tools, others using excel sheets or organizing folders in complex ways, there...lengths to keep accurate records, but given the complexities of modern research, it’s often not enough. A...if they are to switch from paper to digital for example): “… 99% of respondents indicated that ease of ...developed the SciNote Electronic Lab Notebook. Implementing an electronic lab notebook starts with defining...property across various software providers used to supplement their paper notebooks. Instead, all of the important... you’ll be able to do it when needed. How many people from your lab would use the ELN i.e. do you need... facilitate this, some ELNs allow you to invite people and assign them roles and permission that only ...

  49. Antibodies 101: Beyond Surface Labeling

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    Blog Post
    ...lympho-/splenocytes, you might only be interested in one of the various cell populations; for example, CD8...These methods can include labeling markers with multiple proteins/antibodies, targeting markers found within...beyond standard surface labeling. Pro tip! Many people use 'labeling' and 'staining' interchangeably to...antibody can originate from a range of species; for example, rabbit or mouse. In a second step, a fluorophore-conjugated...Otherwise, your secondary antibody will bind to multiple primary antibodies, yielding a false positive ...FACS achieves greater purity of your sorted cell sample than MACS. An effective strategy to sort out your...the permeabilization solution can be the same). Examples of such targets include transcription factors,...

  50. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ...proxy for dimerization (Shaner et al., 2008 - supplementary figure C). However, these assays are qualitative...fluorescent protein. These were either (i) coded by the multiple cloning site and therefore consist of random amino...protein is cleaved by proteases. Nowadays, with ample structural information available, it’s clear that...protein of interest (POI). To indicate the sequence, people often use terminology like “C-terminal fusion,”...#85045). We often use restriction enzymes in the multiple cloning site for generating fusions. For the POI-fluorescent... acids (PVAT, when we use the AgeI site in the multiple cloning site) is sufficient. For the fluorescent... better preserves the function of the POI. For example, the APT1-mVenus fusion localizes at the Golgi,...
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