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We narrowed to 1,010 results for: PLE

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  1. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    ...control of multiple loci is deftly coordinated by the eukaryotic cell for the execution of many complex cellular...endonuclease activity coupled with a customizable single guide RNA (sgRNA) that bears complementarity to a 20bp target...Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR ...toward tinkering with regulatory networks and multiplex gene control. However, these approaches are often...scaffolds to mimic natural transcriptional programs on multiple genes. Modifying the CRISPRi system for multidirectional...scaffold modules are comparatively small allowing multiple sgRNA constructs to be introduced simultaneously... utility of simultaneous and multidirectional multiplex control via scRNA design is readily apparent in...

  2. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...are prone to incomplete silencing of reprogramming transgenes, which leads to incomplete reprogramming... of this method are that it’s relatively simple to implement and doesn’t require time-consuming production...pluripotency, but they also take less time to complete. Morphology is a simple and fast to evaluate. iPSCs should...generating iPSCs. The generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem...vector-free cells Limited genomic integration Multiple transfections required Need to sequence to verify...-Free High High Transgene-free and vector-free Multiple transfections required Protein Delivery DNA-...can complicate comparisons made between lines. Incomplete silencing of transgenes is a concern as well,...

  3. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...generation simpler, as described in an earlier post on generating CRISPR mouse models. For simple knockout...challenging for sequences with complex secondary structure. Today, multiple companies offer long single-...length. There are plenty of Addgene plasmid options for templates - see the example at right using blue.... It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical...certain models remain more difficult to make. For example, large insertions, like a fluorescent protein knock-in...Quadros et al. recommend ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components...guide and your donor ssDNA. Here, we’ll use the example of inserting a 2A-FlpO cassette into the Fgf8 gene...

  4. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...of the promoter as part of a multiple cloning site (MCS). Having multiple sites allows you to easily orient...important that the digest go until completion. The time required for complete digestion varies for different...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...plasmid to the new fragment thereby generating a complete, circular plasmid that can be easily maintained... gene insert with respect to the promoter. For example, let’s say your plasmid backbone looks like the...    Alternatively, this whole process can be completed using a single enzyme if your insert is flanked...gel purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of insert...

  5. The Importance of a Fun Workplace: Company Culture at Addgene

    Type
    Blog Post
    ... Many of these examples we’ve done annually - they are fan favorites! 6. Keep it simple This may be an...anything at all really! We've had plenty of events that included a simple corn hole set and a cooler. Chances...that requires people to be in groups, organize your troops prior to the event, and let people know exactly...power of simple word of mouth - be sure to bring up your event in casual conversation to get people excited...where there is food and beer. Ha! We had over 60 people join the fun - including a lot of future Addgenies...their teammates and project collaborators have completed their work thoroughly. It’s kind of a chicken ...gets much more manageable when you can enjoy the people you spend 40+ hours with each week. In conclusion...

  6. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...template vectors, to perform multiple site mutagenesis and create complex fusion or recombination vectors...Combining multiple Level 2 vectors in the final assembly step permits the creation of more complex constructs...interest does x, but it also participates in y. Depletion of your protein may upregulate an alternative ... recently used by Stephen Royle’s lab, acutely depleted proteins from their compartment of action via ... Sci 2013 May 1;126(Pt 9):2102-13. Assemble multiple component plasmids from building blocks via Golden...Golden GATEway Cloning Frustrated by the complexity of assembling recombination of transgenesis constructs...cloning kit simplifies the cloning process for complex DNA constructs, particular for those involving ...

  7. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...Zika and a closely-related virus, Dengue from multiple sample sources. SHERLOCK could also detect low-frequency... and/or are too expensive, time-consuming, and complex to use outside of standard laboratories. In the...protein variants, Cas13, and Cas12a, to develop simple, portable, and inexpensive platforms to reliably...with crRNA to target an ssRNA of interest, for example, a sequence specific to a virus or pathogen. Once...sensitivity of the assay, targeted DNA or RNA from a sample is first amplified using RPA (recombinase polymerase...reverse transcriptase (RT)-RPA, respectively. RPA is coupled with T7 transcription to convert amplified DNA ...quantitation without compromising sensitivity. To detect multiple targets in one reaction, Cas enzymes, such as ...

  8. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...Before digesting the DNA duplexes with Surveyor nuclease, save some of the duplex and run it as an undigested...products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP duplex) as a control. ...Screening Using CRISPR/Cas9 Deliver Multiple gRNAs at Once - Multiplex Expression of gRNAs Generating Mouse...talks about. After months of troubleshooting, those people who did not tell you that essential thing ask incredulously...recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to target different regions of your gene...reasons to target different regions of the gene with multiple gRNAs, but the most important is that you could...target artifacts by comparing the phenotypes of multiple gRNAs. Targeting different regions of the gene...

  9. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    ... to provide that context yourself. Simple words are not so simple Just as our lab used jargon that required... Addgene move from completing 2-3 large projects a year, on average, to completing 8-10 projects per year... team communication. Although these real world examples did not happen in the lab, they have parallels...words, or words that depend a lot on context. New people in the lab or collaborators will not have the context... that nearly all Addgene teams used that seemed simple, but they could in fact be interpreted in several... the aforementioned lab manager, said the word “sample,” did the word convey the same concept to Morgan... an Addgene-specific definition. Words such as “sample,” “material,” “item,” and “format” all received...

  10. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ...but what if we had a simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR...individual (and multiple) genetic perturbations in single cells. Methods to scale up multiplexed CRISPR systems...was contributed by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts...of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your ...experiments. CombiGEM-CRISPR: The marriage of two simple platforms CRISPR has revolutionized how we decode...mapping the combinatorial genetics that underlie complex regulation in biological systems. The CombiGEM ...products are a diverse pool of lentiviral plasmids. Multiple rounds of digestion and ligation result in plasmid...

  11. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...homology arms (60-70 bp) is added to the complex. This complete transfection mix, i.e. csRNP plus ssODN... Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed with sgRNA...using the csRNP method is as simple as doing routine PCR assays. Here is a simple to-do-list to follow when...immunoprecipitated - along with its associated protein complexes - from the cell-lysate using antibodies against...against the target protein. The captured protein complexes are then analysed by mass spectrometry to identify... protein of interest are pulled-down and later coupled to high-throughput sequencing to identify genome-wide...tool for guide RNA and donor DNA design, and implemented our optimised protocols into a medium-throughput...

  12. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ..., we'll discuss the principles and process of designing a flow panel.  Principles of excitation and emission...Table 1, we can recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE...some background on the color spectrum and the principles of excitation and emission. The visible color...carry their emission peaks within their names, for example, BV421 and RB545. Fluorophores can also come in...en-us/resources/bd-spectrum-viewer.   Another example APC is a "bright" fluorophore when excited by the...building your first panel, we'll talk about one last principle of color emission. As you can see in Figure 1,... of neighboring colors can and do overlap. For example, APC overlaps noticeably with both BV605 and Alexa...

  13. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...your sample to. For example, we use our negative control to compare exposure times of our samples. A prominent...Reproducibility  In order to attain non-bias assessment, multiple people in the lab conduct “independent screening”,...cannot be reproduced and projects that cannot be completed. A 2015 review analyzing and explaining the antibody...Baker, 2015), causing a huge waste of precious samples, time, and funds – up to US$350 million in 2015...techniques on rodent brains and is expanding to more complex brain systems such as non-human primates and humans...noise. We then compare the negative control to a sample with the primary included to confirm that the signal...antibody and not from the background noise. If the sample images similarly to the negative control, that ...

  14. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...insights into the make-up of multiprotein complexes like the EPC1 complex.. Going one step further, they also...employ them in creative and innovative ways. The principle of protein multimerization is seen in many important...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a promoter... localization of a protein via the presence of multiple copies of targeting sequences. Scientists have...This system enabled the specific recruitment of multiple copies of VP64 to the sgRNA-targeted gene, resulting...guide RNAs. Upon stimulation with blue light, the complex recruits a CIB1 binding protein (a portion of CRY2...now take any open reading frame (ORF) and with a simple recombination reaction, generate a protein with...

  15. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    ...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...This complexity index ranges from 0-1, with a value of “0” indicating those markers are completely distinct...stained samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies...the primary detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over ...cytometry relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference...allows the user to use the autofluorescence in a sample as a fluorescent marker. Given that the level of... varies, it is critical to include an unstained sample for each tissue type you are running in your panel...

  16. Hot Plasmids Spring 2024

    Type
    Blog Post
    ...articles.  Here's what you'll find in this post: Multiplexed perturbation and decoding in pooled CRISPR screens...System Neurodegeneration Collection updates   Multiplexed perturbation and decoding in pooled CRISPR screens...screening, Paul Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens...uses two sgRNAs with internal barcodes (iBARs), multiplexed using tRNAs, within the lentiviral 3’ long terminal...reduce recombination and avoid separation of the multiplexed elements. The team designed sgRNAs that include...include an internal barcode and separated multiple sgRNAs with tRNAs, which are cleaved out of the RNA transcript...optimizations produced 90% accurate integration of multiplexed constructs into target genomes and a 10-fold ...

  17. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    ... the ways in which the two guiding principles have been implemented by providing support for connectivity...reproducible, and only the highly restricted number of people who can access the ELN have the ability to attempt... from intensive engagement with a wide range of people at the University of Wisconsin in 2011/12 – researchers...office, and research computing. The two guiding principles emerging from the engagement with the Wisconsin...Reproducibility was both a fundamental driver of these principles and has emerged as a byproduct of connectivity...2012 and 2017 RSpace was developed with these principles in mind, and in response to further input provided...Without such references the record of research is incomplete, making it difficult or impossible to reproduce...

  18. 5 Reasons to Use Reddit for Science Communication

    Type
    Blog Post
    ...just sent people over to r/Science in the initial promotion as a work around… there’s plenty of great ...selling a product than communicating your ideas with people or discussing science, it is a great way to establish...particular hosts a variety of conversations with people who work in many different scientific fields. They...: 1. Reddit has a large and active community of people interested in science. You can see some of the ...of deliberately, cleverly, and secretly pissing people off, usually via the internet, using dialogue)....avoid rousing the trolls. 3. The AMA process was simple and well organized. You can check out the r/Science...work-life negotiation, and mentorship. We can direct people to the AMA in the future and link to it in future...

  19. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ... what makes up your sample size (aka your n value)" and should come from multiple, independent sources...Technical replicates An example of a technical control is the transfection of multiple separate wells (within...experiment from start to finish multiple times and on different days. Using our example above, we would test different...beyond discussion in the abstract and follow an example of a plasmid-based experiment, delineate its requisite...transfected successfully and expressing the protein. For example, fluorescence microscopy images resulting from ...provide the baseline/standard against which other samples can be compared. The Empty Vector Control (mentioned...endogenous gene in your experimental cells. For example, in the experiment above, Plasmid A contains an...

  20. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ... weeks to a couple of months and requires that all of the experiments have been completed. Therefore, ...opportunities in academia - the main reason most people enter postdoctoral training. Similarly, there are...essays in the journals Nature and Science, for example). This blog post is about how to get the most ...a Group Leader position, what will you need? Multiple first or corresponding author publications (published... questions like: What figures are missing? For example, are additional controls necessary? As you build...interesting questions in your own lab. 2. Seek multiple mentors during your postdoc At the end of grad...Discussions can reveal mutual scientific interests, complementary perspectives, a shared communication style. ...

  21. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    ...remaining letters should be from independent people – i.e. people who have not worked with you directly or... connect with people who could be beneficial during a career search. Doing these simple things will make...refine and build skills overtime for success. Many people will have different roles, titles, and responsibilities... to find the perfect job immediately after you complete your Ph.D. or postdoc. Foreign national researchers...standard of proving that you are one of the top people in your field. No employer is necessary and no ...to the field that have had a major impact. For example, you were the first person to identify a novel ...successes in the performing arts- not relevant at all. Please do not include the fact that you are a classically...

  22. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...assembly, assembling fragments in multiple steps to avoid using multiple fragments that share homology in...overhangs perfectly match up. They just need enough complementary ssDNA to hold the plasmid together, even if ...if there are gaps or flaps remaining. This incomplete plasmid looks like a homologous recombination intermediate...to work fixing up the “damage” and producing a complete plasmid. Besides being compatible with any vector...either by annealing a mixture of products or from incomplete PCR — although higher DNA concentrations are ... separate PCR products (middle branch); or by incomplete PCR, which skips the final extension step to ...single-stranded overhangs (right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product...

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

    Type
    Blog Post
    ...of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and upon binding, gets activated...the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity ...strip. Highlights: Of the 83 known COVID-positive samples tested, DETECTR has a positive prediction rate ...°C water bath Approach: RNA is extracted from a sample and subject to isothermal recombinase polymerase...presence of two or more mismatches in the cRNA:target duplex and will not give a positive result for highly ... can be performed in two steps for challenging samples as in Two-Step-SHERLOCK or in a fast, high-throughput...incubated at one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the...

  24. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...mutational scanning to simultaneously test how multiple amino acid changes affect a protein of interest...and easy technique that can be used to generate complex libraries of mutant plasmids ready for deep mutational...a selection system. You can test the effect of multiple mutations in combination and possibly discover...in protein activity. See Natarajan et al for an example of this. Plasmid library generation prior to one...mutations are introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil...second universal primer is used to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled...uracils that are in DNA and ExoIII recognizes nicked duplexed DNA. This results in the final product having ...

  25. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...This allows for the multiplexing of sgRNAs against unique genomic targets or multiple sgRNAs against the...enzymes (Weber et al., 2011). It can be performed at multiple levels to generate anything from individual transcription... favorite gene-of-interest, all the way to the complex multi-TU constructs that compose genetic circuits...assembly can occur. It is quite extensive with a supplemental add-on kit and is compatible with E. coli and...modules, and many more of the usual suspects. The Multiplex Yeast Toolkit builds on the YTK and adds several...capabilities, including conditional gene expression, multiplexed CRISPR, and chromosomal integration of larger...intuitive CRISPR MoClo application is to assemble multiple unique sgRNAs into a multi-TU targeting construct...

  26. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ... exact amount of protein in your sample, you’ll compare your samples to a standard curve. This curve is...the known samples is used to calculate the concentration of protein in the experimental samples. Get more...expression of multiple proteins on the same cell simultaneously as well as compare between samples. Learn more...heavy chains, and the rest of the Fab regions are completed with the Ig light chains. The variable region ... protein because the antibodies will recognize multiple epitopes on the protein. However, each animal ...optimized for specificity and sensitivity, for example by changing certain amino acids to improve its ...expression in cell lysates, whole cells, or tissue samples. Many experiments that use antibodies use a primary...

  27. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ...the plasmid complements the deletion). Find the Plasmids Here Plasmid features enable multiple student experiments...abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is corrected,..., well-studied and inexpensive. In addition, a complete set of well-annotated deletion strains is available...occurs through a well-characterized pathway with multiple enzymatic steps (5). In their experiments, students...ura3 mutants and cannot grow without uracil supplementation (6). Expression of MET/Met genes from the pYES2.1...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...

  28. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    ...activation experiments. In cases of multiplex gene regulation (activating multiple genes at once), however, SAM... peptide array to fused with multiple copies of VP64. By having multiple copies of VP64 at each loci of...VPR fuses a tripartite complex with dCas9 to activate transcription. This complex consists of the VP64 ... of CRISPR/Cas systems, gene activation across multiple loci was an arduous process. When using zinc finger...sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest. Upon binding, CRISPRa...upon the initial dCas9-VP64 method, so there are multiple options to choose from when looking to activate...CRISPR activator. While it requires a relatively simple construct, it exhibits modest levels of gene activation...

  29. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE (Multiplex Automated.... Adding complexity, they found that they could also include a CRISPRi system for multiplexed modulation...functions in S. pneumoniae and can be used to generate multiple mutations simultaneously (Jiang et al. 2013). ...mutations and 66% efficiency for small insertions. Multiplexing of two targets simultaneously is possible with...pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing...multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression (CRISPRi) in bacteria...transcription by physically blocking the elongation complex from binding the DNA or extending the transcript...

  30. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ... The simple answer is that sometimes it is necessary or desirable to control or look at multiple genes...together for complex genetic analysis in Drosophila or for precise control over multiple genes in synthetic...binary systems. In recent years, a number of more complex systems have also been developed which don’t fit...that transcription is now OFF.   We’ll look at examples of both natural and engineered repressible promoters...transcriptional activation domain VP16 taken from the herpes simplex virus. tTA binds to promoters containing TetO elements...in mammalian cells, this system has also been implemented in a variety of other organisms, such as sphingomonads...UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4...

  31. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors...systems work using a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade...compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade is an important... Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex, developing... settings — mostly a consequence of Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR...component of the CRISPR Cas3 system. Most Cascade complexes are comprised of a combination of Cas5, Cas6, .... It was originally suspected that Cas3 moved a couple of kilobases down the strand of DNA, Cascade caught...

  32. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

    Type
    Blog Post
    ...production site to its final destination. As an example, most secreted proteins are produced in the endoplasmid... the untagged, wild-type protein. You may, for example, validate that the fusion protein localizes properly...FPs to highlight various subcellular structures. Please see Figure 1 for commonly used markers for major...fusions to visualize subcellular structures. For example, you might use your fluoresent protein fused to...and potentially which other proteins it forms complexes with. These “colocalization” studies give insights...within the same subcellular structure or protein complex. By coexpressing both the marker protein and the...possible to determine the location of proteins within complex structures. In order to interpret colocalization...

  33. Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

    Type
    Blog Post
    ...constructs to allow people to, for example, knock out a gene with simultaneous complementation, with a knockout...enable other people to do that too. We don’t have the capacity to send tools out to people ourselves, but... but we’d really like for people to use them. The MoClo Plant Kit makes it really easy to clone genes ...but have comparable expression levels to things people do use a lot. The problem in plant science is there...similar stability to transcripts as the ones that people use all the time. Now there are many, so if you... those working in plants, including many things people already use. It makes the switch to Golden Gate... Golden Gate is a conceptual barrier. Sometimes people don’t know how to start. They might want to use...

  34. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    ...process such as primer and plasmid design. Examples:  “multiplex design”, “multi-method design”, and “single-method...deals in post-experiment quality control, a good example is CRISPR-GA. These tools assess repair events ...published 1 Jan 2013, no longer available), is a simple catalogue of reported sgRNA designs. The second...similar to EENdb than CrisprGE, but is also a good example of how database tools can be used to aid design...this category are the most common between tools. Examples: “single-target design”, “multi-target design”...considered extremely useful, depending on design goals. Examples:  “feature aware”, “SNP aware”, “secondary structure... common utility functions are batch design or multiplex features. However, tools aiding in primer design...

  35. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ... as the building block of our understanding of complex biological processes. Many tools exist to perturb...in the context of the biological question. For example, while gene knockouts have long been considered...silencing,” of any gene that contains sequence complementary to the dsRNA (Wilson and Doudna, 2013). RNAi... translation, or chromatin modifications of complementary gene sequences (Figure 1). As a result, gene... of interfering RNAs.   RNAi vs. CRISPR: Complementary techniques It is fair to say that CRISPR has...of the technique—all that is required dsRNA complementary to your gene or genes of interest to trigger...constructs that enable insertion of ~400 bp complementary to your gene target as inverted repeats (Figure...

  36. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...translational, biomedical, or applied sciences), people conduct research to answer questions about how ...applied. The current pandemic serves as a striking example of the applicability and importance of research...you’ll see in a lab. Position Common tasks Principle investigator (PI) Runs the lab Writes a lot ...questions and your excitement will in turn inspire the people around you. Be proactive Rather than applying only...the best research experiences typically come when people actively seek out science that genuinely excites...experience more tailored to personal interests. Talk to people, ask questions, and dig deep Talk to your professors...into is really important.  As you’re talking to people, though, try to be conscious of who has your best...

  37. Viral Production at Addgene

    Type
    Blog Post
    ...single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR. Human... lot. At Addgene, AAV particles are titered by droplet digital PCR (ddPCR) using primers and probes targeting...specific QC experiments were performed on your lot, please contact us. Full sequencing of the Viral Genome...of the viral capsid proteins VP1, VP2, and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample.   Sterility Viral vector preparations are ...Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified...

  38. 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...

  39. A Quick Guide to a Career in Software Product Management

    Type
    Blog Post
    ...always tell people the best part of my job is getting to talk to so many different people! I communicate...skills: working with data, working with people, working with complex processes Arm yourself with knowledge...management, and how does it relate to science? In the simplest of terms, a software product manager helps ensure...group is building the software tools for the right people at the right time. To explain a bit further, a ...manager works with software developers to help implement solutions for the stakeholders. Finally, software...all fits in with science. Well, I’ll give you an example. At my previous company, Ginkgo Bioworks, our software...backgrounds, most did not. It was my job to translate complex lab operations workflows into a format the developers...

  40. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...splitting up a sample into thousands of physically isolated partitions (such as droplets or wells on a ...clones for complete functional knockout of your target gene. Mismatch nuclease assays are completely “blind...of target DNA molecules present in the original sample with a great deal of precision. This means digital...theoretically detect a single target molecule in a sample. Digital PCR assays for mutation screening Now...use aren’t that much different from conventional duplexed primer probe assays often used in real time PCR.... The drop-off probe is designed to perfectly complement the wild type sequence and is not capable of ...amount of both mutated and wild type target in each sample can be quickly calculated (details in [2]) and ...

  41. 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...

  42. 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...

  43. 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 ...

  44. 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...

  45. 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...

  46. 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...

  47. 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...

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

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    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...

  49. SciComm: Taking Science to Elementary and Middle Schools

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    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...

  50. 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...
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