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  1. Science Career Options

    Type
    Blog Post
    ...critically and carry out in-depth research is applicable to a wide range of industries and is a highly...skills acquired from a science degree that can be applied to many roles. These types of skills show that ...sectors such as forensics currently have too many applicants compared to the number of available roles). Make... Make your resume stand out Before you begin to apply for a job, it is useful to search online job websites...your research, it’s time to start sending out applications. Check out our other career posts for more specifics...careers and to find advice on crafting the perfect application. Additional Resources on the Addgene Blog Learn...

  2. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    ...   Limitations of the in silico approach The in silico approach cannot replicate the complexity of ...Better together Combining in silico and in vitro approaches enables researchers to develop safer and more...unwanted immunogenicity. The 3-D models can be applied to corroborate the occurrence and extent of the... somewhat act like a 2-step verification. All approaches have their own limitations. Using methods together...together can inform decisions about in vivo application and clinical testing. This would pave the way for...

  3. Management for Scientists: Delegating is Key

    Type
    Blog Post
    ...dramatically improved when tasks are distributed appropriately. If team members have opportunities to develop...develop and stretch then they will be happier and more productive in the long run.  If you are overwhelmed...the copy, design every icon, curate all of the appropriate plasmids, and code the entire page. That would... page. The team manager must then facilitate appropriate communication between members of the team to ...some important criteria for delegating tasks appropriately: Are the desired goals and outcomes clear? ...questions. If you are perceived as too busy to be approachable mistakes will be made that could have been prevented...

  4. AAV Q&A with Tim Miles

    Type
    Blog Post
    ... our knowledge. Disease Applications Q13: Does AAV have potential applications in diseases which may be... be best served by a dual vector, split intein approach.   Q3: What’s the best method for titering AAV...1x1014 vg/ml.. As evident in our repository, this application is an outlier for PHP.eB (1x1011 - 1x1012 vg/...systemic injections? A: We have not observed any appreciable difference in AAV transduction efficiency between...CRISPR tools is an exciting area for future AAV application to epigenetic editing. This area is discussed...

  5. Hot Plasmids - February 2022

    Type
    Blog Post
    ...editing applications.  Figure 4: Overview of CasMINI for genome engineering applications. Image ...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...challenges for in vivo delivery and cell engineering applications. CasMINI, a new miniature CRISPR-Cas system ...

  6. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    ...cell type doesn’t always lend itself to direct application of virus. However, the cargo capacity of AAV ...gamma-retro viruses. This is large enough for many applications, but in some cases multiple AAVs (co-infection...and AAV. If you’re wondering more about what applications these viruses are good for, we have plenty of...Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New...Gammaretroviral vectors: biology, technology and application. Viruses. 2011 Jun;3(6):677-713. DOI: 10.3390... Campos MD, Materatski P. An Overview of the Application of Viruses to Biotechnology. Viruses. 2021 Oct...

  7. How to Keep a Lab Notebook for Bioinformatic Analyses

    Type
    Blog Post
    ...information in the future. Approach: Outline a brief overview of the approach that you will take to help...biologists are finding themselves using computational approaches to analyze large data sets (myself included) ...your analysis, even if your conclusion is “this approach is not ideal because…” Including a conclusion ...or may not be where you also record your goal, approach, and conclusions (see above). Keep original ...in my own research. Again, some of this may not apply or may not work for you. The best way to find a ...

  8. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...fluorophores, together, are able to give you the appropriate readout. A common method of labeling proteins... wavelength. Likewise, the emitted photons also appear within a range of wavelengths. For that reason,...excitation and emission are the most efficient (i.e., happen most of the time). Figure 1 shows a graph from ...higher-wavelength emission spectrum compared to APC. This happens due to a process called Fluorescence Resonance ... the fluorophore, and whether that fluorophore appears alone or in conjugation with another fluorophore...absorption at 650 nm, APC-Cy7's optimal excitation happens at higher wavelengths of about 750 nm. Source: ...Besides some background cell death constantly happening in cell culture, the transduction process itself...

  9. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    ...upcoming posts in this optogenetics primer series, I’m happy to share some of the tips and tricks and I’ve learned...memory task, on the other hand, would not be an appropriate test.  Of course, proper controls are required...using these lines is, if one is crossed with an appropriate mouse line expressing a particular cre, viral...22430017. 2. Fenno L, et al. The development and application of optogenetics. Annual review of neuroscience...PMC3225502. 4. Mattis J, et al. Principles for applying optogenetic tools derived from direct comparative...neuronal populations using combinatorial viral approaches. Current protocols in neuroscience / editorial...PMC3191888. 9. Zeng H, Madisen L. Mouse transgenic approaches in optogenetics. Progress in brain research. ...

  10. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    ...developed by the James and Schuh labs. Clift et al. applied TRIM-away to nine proteins in ten ddifferent cell... sometimes incorporating chemically inducible approaches to provide temporal control, this new system,...Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid and reversible recruitment of...target multiple loci at once. In addition, the approach allows minute-by-minute analysis of the kinetics...domain. Transcription factor activation via the appropriate ligand - estradiol for hER or progesterone for...leads to nuclear localization, binding to the appropriate promoter sequence and activation of gene expression...

  11. Meet an Addgenie: Insights into Grad School, Career Paths, and More

    Type
    Blog Post
    ...the co-op application process, my advisor encouraged me to keep my values in mind as I applied. Thinking...My name is Lauren Flynn and I am currently wrapping up my co-op with Addgene as a DNA Sequencing Analyst...s and leave. I was really devastated when that happened. I didn't know if I should even stay in science...in your career path and still find comfort and happiness there. Read all of the Meet an Addgenies features...connections with my coworkers that may not have happened if we hadn’t had these conversations. Now, I am...

  12. Synthetic promoter AAVs for cell-type specific expression in retinal cells

    Type
    Blog Post
    ... Ophthalmology Basel recently took a different approach to enhance specificity in retinal cells. To do...type specific expression in the retina.  Four approaches for designing synthetic promoters  To build a...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry... rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling would...

  13. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    ...simpler, yet more effective, CRISPR-based epigenetic approach (spoiler alert: it’s dCas9… with no modifications...). But there are a number of drawbacks to this approach: TET proteins are not demethylases; they are ...and Szyf 2021).    Figure 3: A simpler approach - dCas9 blocks DNMT1 methylation, leaving the ...resistance genes.  It’s that simple! Good luck and happy editing!    Thank you to our guest blogger!Daniel...Addgene blog CRISPR101: Editing the Epigenome  Mapping the 4D Nucleome with CRISPR/Cas9 Resources at ...Xu X, Tao Y, Gao X et al. (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discov 2,...

  14. 5 Tips for Troubleshooting Viral Transductions

    Type
    Blog Post
    ... to increase AAV transduction efficiency by approximately 15-fold (Chen et al., 2006). 3. Transduction... at 4 °C). After ultracentrifugation, virus may appear as a white pellet. The pellet can then be rinsed... This antibiotic selection can be performed approximately 72 hours after transfection and should give ...give rise to a surviving population of approximately 20–50% of the packaging cells after 1–3 days of selection... could be examined with a microscope and should appear fluorescent if they were infected. While there ...transfection, higher-level expression after approximately 72 hours is likely the result of successful ...

  15. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...to be less expensive than an ELISA kit. In this approach, viral RNA is first converted to cDNA and then...several different lentiviruses in parallel, this approach should be considered. Similar to the p24 assay... Fluoresence Titering Protocol Another popular approach utilizes the antibiotic resistance genes carried...have integrated into a target cell’s genome. This approach can discriminate multiple integration events, ...underestimating the viral titer. The main drawback of this approach is that it can be quite labor intensive. Target...virus may want to choose more straightforward approaches such as the FACS-based or colony-forming unit...

  16. Academic vs. Industry Postdocs

    Type
    Blog Post
    ...your resume pulled to the top of the pile when you apply for such positions. Of course, if your goal is to...transition from an academic setting—especially if job applications require teaching experience or high numbers ...web site. Each position is for a specific, pre-approved project. Other companies, such as Novartis, have...s program. Once you’ve found a few positions to apply for, compare and contrast industry postdocs at those...not. The point is that each company has a unique approach, and some might suit your needs better than others...background in cell and developmental biology and is applying this towards researching neurodegenerative diseases...

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

    Type
    Blog Post
    ...10 modules often rely on two-step hierarchical approaches using different Type IIS restriction enzyme specificities...transformation plate can be manipulated to result in appropriate levels of colony plating densities. Ligase fidelity...accuracy of assembly. The fidelity data can be applied to derive similar high-fidelity overhangs for any...researchers continue to test the limits of DNA assembly approaches with increasingly complex experimental conditions...Kucera is a Principal Development Scientist of Applications & Product Development focusing on DNA cloning...Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias...

  18. Of Myc and Men

    Type
    Blog Post
    ...The strategy they landed on was to recombinantly append their proteins of interest with unrelated peptides...generate a reliable antibody for protein tagging approaches” was not part of the goal when Evan’s team generated...plays in human health and disease. But no science happens in a vacuum!  Munro and Pelham learned of these... flexible - it could now be used in way more applications.   Fig. 1 Making a Protein Tag (Abridged...their study, some of the newer Myc-tag antibodies appeared to be less sensitive to sequence context.    So...proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70. Embo J...

  19. Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

    Type
    Blog Post
    ...18% to 65% (Maggio et al., 2014). An alternative approach to NHEJ is to utilize the Homology Directed Repair...phenomenon to the presence of terminal proteins capping the ends of linear double-stranded AdV genomes....double-stranded chromosomal DNA breaks “that always happen naturally.” These adenoviral CRISPR/Cas9 genome...and quiescent, post-mitotic mammalian cells. Applications of CRISPR delivery by adenoviral vectors Since... Adv vector delivery is often used, the CRISPR approach based on HDR has the advantage of being a potentially...Strategies for Tissue-Specific Targeting. In: Applications of viruses for cancer therapy. Elsevier, pp ...

  20. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ...and Mar. 1, 2020. Figure 2: A simple approximation of the effect of COVID-19 on data smoothed with...a 7-day rolling mean. While this global approximation already shows us a rough "COVID-19 effect" on...maximum likelihood estimates of each parameter. We approximated the data likelihood with multivariate Gauss-...the supposed end of lockdown), request activity appears to return to normal. The model fit these two discrete...is a Ph.D. candidate at MIT. His research uses applied machine learning and statistical methods for fundamental...fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate student at Harvard...

  21. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...involves partitioning a PCR reaction mixture into approximately 20,000 droplets using water-oil emulsion technology...to create a water-in-oil emulsion containing approximately 20,000 droplets. Each droplet contains the material...droplets. In the image to the right, there is approximately 1 positive copy per microliter in the no template... use ddPCR for AAV titration, there are many applications for the technology.  Droplet digital PCR is ...being used in the detection of microbes. Some applications include measuring viable probiotics and detecting...detecting circulating pathogens.  With its many applications and ease of use, droplet digital PCR is becoming...

  22. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    ...a number of labs are using enChIP, and I'm very happy to see that many researchers all over the world ...describe this technology and some of its potential applications? Fujii: We generated a series of retroviral ...the peer-review system of journals and grant applications, for example. However, at the same time, it'...Throughout your career, you have held research appointments in the US (New York University School of Medicine...which only one was done in Japan, which is very disappointing. I hope I can help change the situation in the...and graduate students as they complete their appointments in your lab?  Fujii: These days it is very difficult...

  23. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

    Type
    Blog Post
    ...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...Re-engineering GFP to increase its range of color and application Almost as soon as its sequence was elucidated...the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other...change. Typically, two fluorescent proteins with overlapping excitation/emission spectra are used; one fused... just scratched the surface of the potential applications for GFP. It has also been used to identify particular...

  24. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...hour. Find the SHERLOCK plasmids at Addgene! Applications of the SHERLOCK detection system The Zhang lab...portability. This method can be used for an array of applications including clinical diagnostics (e.g. pathogen...While the lab points out that the protocol is not approved for clinical use at this point, they hope that...patient samples. Find the DETECTR plasmids here! Applications of DETECTR in diagnostics DETECTR allows for...an array of molecular and clinical diagnostic applications. The company Mammoth Biosciences has been started...SHERLOCK detection system, DETECTR has not been approved for diagnostics yet, but is undergoing validation...

  25. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...This distinct approach means researchers can now select the best tool for their application from among many...incorporate A bases across from abasic sites. This approach resulted in a maximum A → T product purity of ...AYBEv3!). Although AYBE, AXBE, and ACBE do not approach the high product purity of CBEs and ABEs, they...). Several different teams succeeded with this approach around the same time (He et al., 2024; Tong et...on in the base editing field! From CRISPR-free approaches to mitochondrial base editing to base editors...

  26. Hot Plasmids: Fall 2024

    Type
    Blog Post
    ...performs well in many different antibody-based applications.  IPI’s recombinant version is a fusion of mouse...ligase cereblon (CRBN), including popular new approaches like PROTAC targeted protein degraders. But CRBN.... elegans embryo, where developmental processes happen within a few hours. In order to use the latest ...cellular processes in C. elegans: mScarlet3 for applications needing intense brightness (such as low-expression...simplicity and efficiency of this dual-plasmid approach makes it a valuable resource for advancing our...when the module unfolds under tension. When this happens, BiT and the LgBiT luciferase fragment (expressed...

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

    Type
    Blog Post
    ...difficult to source a commercially available and appropriately conjugated antibody. While labs can conjugate...find,  it is often easier for labs to source appropriate antibodies for an indirect ELISA than for a direct... capture antibody must bind to distinct, non-overlapping epitopes so that they do not interfere with each...specific and can be difficult to develop if an appropriate conjugated antibody is not readily available....cross-reactivityChallenging to develop assay Competitive Appropriate for small moleculesReduced chance of cross-reactivity...choose the best ELISA for your needs. Good luck and happy binding! More resources on the Addgene blog Antibodies...

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

    Type
    Blog Post
    ...cells retain the same color, allowing for the fate mapping of cell populations over time. The ability to track...variety of species, cell types, and research applications. Labeling neurons with Brainbow The Lichtman...neurons in the cerebellum of mice, the team appropriately named their technology Brainbow. Brainbow 1.0...level of these XFP is not strong enough for all applications. To resolve this, similar systems have been ...cell type when crossed with mice expressing the appropriate Cre allele. The Confetti mouse contains the Brainbow...PMC3713494. Ghigo, Clément, et al. "Multicolor fate mapping of Langerhans cell homeostasis." Journal of Experimental...

  29. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ...Depending on the research question and downstream application, one or several of those techniques can be used...your flow analysis. However, certain downstream applications, like an in vivo adoptive transfer of those ...or free amines of proteins. Depending on the application, they can be membrane-permeant or membrane-impermeant...are positive or negative for the dye. The main applications of dye stains in flow cytometry are cell proliferation...with a high resolution for specific downstream applications, while maintaining a clean cell surface, dump...and bind DNA or free amines, depending on the application. Paul Heisig is a Research Associate in the lab...

  30. Viral Vectors 101: AAV Serotypes and Tissue Tropism

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    Blog Post
    ...experimental system. AAV1 AAV1 was the first viral vector approved for gene therapy (Issa et al., 2023). It may be...vessel barriers. It is especially popular for applications targeting liver tissue. In addition, it has ...the blood-brain barrier, making it useful for applications targeting the CNS (Issa et al., 2023). Additionally... 2016). This functionality makes it useful for mapping neural connections. Although derived from AAV2,...treatment, which can pose problems for gene therapy applications. Cross-reactivity of AAV serotypes varies by.... A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), ...

  31. A Control for All Seasons

    Type
    Blog Post
    ...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding... unavailable, try substituting with knockdown approaches using siRNA or shRNA. If this alternative is ... alternative, albeit less ideal, verification approaches discussed in the Antibodies 101: Validation post...Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity must also be ...

  32. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...you with these, so keep reading! Good luck, and happy optimizing!  Recommended Reading Issa, S. S., Shaimardanova.... A. (2023). Various AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), ...A. (2021). Next Step in Gene Delivery: Modern Approaches and Further Perspectives of AAV Tropism Modification...AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways...Retrograde Serotype Viral Vectors 101: Viral Applications...

  33. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    ...making it very useful for genetic engineering approaches. Most importantly for this technology, however...piggyBac™ technology can be used for numerous applications, including transgenesis, gene-trap screens, ...ssODN) as the donor [6]. The problem with this approach is that, since there is no selection, you will...technology that may be used for a variety of research applications, including making transgenic cell lines and ..., while avoiding the pitfalls common to other approaches. Many thanks to our Guest Bloggers Amar Singh...Fingers, TALENs, and CRISPR-Cas9 nucleases. Mathew applies his specialized knowledge to help researchers identify...

  34. Why Do I Need an MTA Anyway?

    Type
    Blog Post
    ...created by the Recipient Right to file patent applications on modifications or inventions Right to publish...can even sign electronically or choose to auto-approve MTAs. Whatever your preference, we are here to ...to make it fast and easy. Fast Approval: The UBMTA used by Addgene is internationally recognized as...transfer officers are familiar with the UBMTA and are happy to agree to it. Their confidence and familiarity...familiarity with the UBMTA result in quick approvals, so your plasmid requests can get to your lab faster. For...

  35. In Living Color: The Skinny on In Vivo Imaging Tools

    Type
    Blog Post
    ...have used fluorescent proteins to observe what’s happening inside cells for at least a couple of decades....of hues. While those tools are great for many applications, Verkhusha and his lab at Albert Einstein College...exactly what they reported doing in a paper that appeared in Nature Biotechnology back in 2011 and then ...Verkhusha’s team reported another advance in the application of near-infrared fluorescent proteins, iRFP670...contrast agents for two-color imaging in animals. The approach relies on ultrasound waves as opposed to light...

  36. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    ...increase specificity. This modular approach made ZFNs highly appealing to researchers.  TALENs In 2010, ...fiction, was also a trained biologist. This is apparent in his work The Island of Dr. Moreau (1896) where...made SciFi director Ridley Scott wonder what would happen if these corporations would extrapolate genetic...successful in the box office, indicating the insatiable appetite of the moviegoers for dinosaurs wreaking havoc...also transcended into the world of SciFi. This is apparent in the use of the word ‘CRISPR’ for anything associated...

  37. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ... Normal Variables – Independent Variance – Approximately equal Data type - Quantitative If your data ...null hypotheses. Benjamini uses a complementary approach which identifies a p value cut off. This method...all generally yield a similar result and are appropriate to use if all sample comparison is needed, without...keep your friendly neighborhood biostatistician happy by making your statistical analysis plan when you...right statistical test, just do your homework and happy testing!   References Wissing, Dennis R., and Timm...Variables: A Monte Carlo Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389/fams...

  38. Chemogenetics vs. Optogenetics: Which Method Should I Choose?

    Type
    Blog Post
    ...beneficial for use in therapeutic or behavioral applications where drawn out effects are required. Targeted... subcellular regions of a neuron. Optogenetic approaches excel at discrete cell or tissue-subregion targeting...to the limits of light delivery. Chemogenetic approaches excel at broad tissue or system-wide targeting...chemogenetics. Note that, as with all tools, both approaches suffer from unique sets of off-target side-effects...EndogenousNot easily controlled Unique advantages Mapping the brainDissecting circuits Studying GPCR pathways...

  39. Hot Plasmids and Viral Preps - July 2021

    Type
    Blog Post
    ...users have recently begun to develop CRISPR-based approaches for studying gene function in these systems. ...use in a fetal-lung derived organoid system, the approach is intended to be amenable to organoids derived...tissue-types. By bringing together CRISPR-based approaches and organoid models, toolkits like this one provide...trigger seed germination of parasitic plants, this approach was also used to build and validate a ratiometric...parasitization of cereal crops. As a result, agricultural applications of these novel biosensors could have a major...

  40. Some Like it Hot: Thermostable GeoCas9

    Type
    Blog Post
    ...thermophiles, bacteria must be grown at lower temps. This approach only works for facultative thermophiles (high ... occurs? This is different than the engineered approach used to make eSpCas9 and SpCas9-HF1. By mining...SpCas9 RNP’s and a guide targeting a site with an overlapping PAM. In both cases, GeoCas9 and SpyCas had similar...could also be used with in vitro molecular bio applications that require high temperature reaction conditions...always, the plasmids are available from Addgene. Happy CRISPRing! References Harrington, Lucas, et al....

  41. Antibodies 101: Affinity Tags

    Type
    Blog Post
    ...tag to remain on your protein for downstream applications Below, we describe a few of the most common...not to perturb protein function for downstream applications. Tag cleavage must be performed after primary...also provides a visual detection mechanism via applications such as western blot and immunofluorescence....dimerizes in solution, which can affect downstream applications. Also, purifying GST by affinity chromatography... are generally best suited for their primary application. Ready to start tagging? We are here to help ...

  42. Tips for Improving Your Next Manuscript

    Type
    Blog Post
    ...scientific aspects of writing. Following his advice, I applied to the class. Receiving the email that I had been...the ASM headquarters. I decided to go ahead and apply; thinking I probably would not get selected for ...knowledge with practical understanding of how to apply it. The personal interaction of critiquing others...well as being critiqued added an extra level of application not present in the online class.   Four ways ... manuscript should be able to be enjoyed and appreciated by all fields not solely the specific area the...

  43. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    ...learn how easy this process is and to get tips for applying it in your own lab. Table 1: Comparison Between... E. coli and transformants selected with the appropriate antibiotic. Although the precise mechanistic ...additional 15-17 bases that are complementary to the overlapping vector ends. See an overview of the PCR process...an agarose gel. You should see bands with the appropriate sizes for your vector and insert. Once you’ve...correct product. Once you find a colony with the appropriately sized product, purify and sequence the plasmid...

  44. What the HEK?

    Type
    Blog Post
    ...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first...cell biology, there are thankfully many other applications that they are useful for! What the HEK is it...cell line and we will highlight some of those applications here.   Virus production HEKs are the go-to ...proteins at high levels. In a case where clinical applications of recombinant proteins are necessary, HEKs ...significantly reduces the cost of production. Research applications There are two areas of research where HEK cells...

  45. Comparing Cas9 to NgAgo: Can the Argonautes Best CRISPR?

    Type
    Blog Post
    ...NgAgo replace CRISPR? Such a drastic shift won’t happen overnight, but there are a few reasons why you ...flexibility in choosing a genomic target. NgAgo also appears to outperform Cas9 in GC-rich regions. DNA guides...staggered (Cpf1) cleavage. NgAgo takes a different approach, randomly removing 1-20 nucleotides from the cleavage...site specified by the gDNA. Depending on your application, this could be favorable or unfavorable. In a...activity. One-guide faithful NgAgo guide loading appears limited to the period when the protein is being...

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

    Type
    Blog Post
    ...generation of various flux imbalances. The conventional approach has typically been to either systematically delete...networks and multiplex gene control. However, these approaches are often met with the challenges of regulatory... possible. From a design perspective, this is appealing since the RNA scaffold modules are comparatively... multiplex control via scRNA design is readily apparent in metabolic engineering. The authors elegantly...control of gene expression. This strategy can be applied to effectively change metabolic flux. The dCas9...

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

    Type
    Blog Post
    ...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the pull-down...disrupts protein function, an N-terminus tagging approach should be pursued, however, clonal lines must ...me at [email protected]. Keep pipetting and happy genome editing! Pooran Dewari is a postdoc in Steve...optimizing CRISPR/Cas9 genome-editing methods and applying them to study brain tumour biology. When not pipetting...

  48. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    ...axial dimension that will appear in focus). This means that the images will appear sharp without interference...excite a molecule of GFP. Because red light has approximately half the energy of blue light, two photons of...density of red-photons be establish for this to happen.Using red light has a second advantage: red light ...objects lying within the diffraction limit will appear as one object in the final image. Over the past...of light – think of a single GFP molecule – will appear as a fuzzy 200-300 nm spot when imaged. Super-resolution...

  49. Fluorescent Protein Travel Awards - FLiPs and Fluorescent Protein Biosensors

    Type
    Blog Post
    ...interactions. Beyond these common applications, researchers are finding new ways to apply fluorescent proteins everyday...Zhou and Fatima Enam. There were many excellent applicants but Xin and Fatima stood out for their creativity...Light-inducible Proteins (FLiPs) Fluorescent proteins can be applied in a long list of creative ways. In her graduate...utility of these FLiPs in a variety of research applications and particularly notes their ability to be precisely...

  50. Hot Plasmids - January 2023

    Type
    Blog Post
    ...have high levels of off-target effects, both of happen with Cas13. CRISPR-Csm degrades both nuclear and...antibodies may just not perform as well in one application as another, so it is always best to have options...for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page for more...fluorescent proteins (FPs) necessary for many of these applications have lagged. Fixation, chemical staining, and...well-suited for the demands of advanced microscopy applications such as super resolution, electron, and expansion...
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