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Showing: 211 - 240 of 884 results

  1. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    Published
    Sept. 24, 2019, 12:55 p.m.
    ...colonies, check that the antibiotic in the plate correspond to the antibiotic resistance marker present...hopefully obtain your coveted plasmid with no substantial delays. Design, design, design Before diving ...fragments. You can generate the parts of DNA that you want to assemble together in a combination of different...difficulty amplifying your target sequence, or if you want to amplify sequences from a large genome organisms...successful and ultimately faster cloning. Remember to quantify the DNA concentration of all the samples by gel... experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check the plates...E. coli  strain known to be sensitive to that antibiotic, and if the strain is growing fine, it is probably...

  2. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
    Published
    June 15, 2017, 2:30 p.m.
    ...between studies is substantial (e.g, the poor performance of mRuby2 is detected by Constantini et al (2015) ...steadily increasing. Of course, you want the ‘latest and greatest’ variants with the best properties for your...dimerization tendency, as is present in EGFP. Constantini et al, (2012) developed a cell-based assay that...If homodimerization occurs (most likely in an antiparallel configuration), typical structures known as ...OSER) whorls are visible. These whorls can be quantified for individual cells, and the number of 'normal...tagRFP does not act as a true monomer in cells (Constantini, 2012), whereas it was previously concluded to..., good performance in the OSER assay is not a guarantee that using this fluorescent protein to tag your...

  3. A Guide to Designing a Scientific Poster: Content, Layout, and More

    Type
    Blog Post
    Published
    Oct. 22, 2019, 12:20 p.m.
    ...only the most important results to your audience. With a poster, you have the advantage of being able ... sections as a primary research article, it’s important to understand that presenting your work in poster...certain posters resonate with you (or not). It is important to reflect on these impressions when thinking ...present at your poster at all times, so other participants can identify you. You may also wish to make ...particular styles recognized to work well. It is important that your readers can navigate your poster and...and acknowledgements sections are inserted. The advantage of this style is that it will display your data...pattern across the poster. This style has the advantage of being different and potentially more catchy...

  4. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ...sequence are important considerations for designing your gRNAs. For indels, it's not so important what location...nom. Me love gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment ...tool among them. While this seems obvious, it is important to remember that the same is true when designing...location in the gene you target, but it is important that your gRNA sequence is designed to be highly active...from). Finally, for HDR, location is much more important because you have to target within ~30 nt of your...to the intended edit rather than some passenger variant that came along with the single cell clone (although...SaCas9, NmeCas9, Cas12a enzymes, and engineered variants thereof offers additional PAM options that can...

  5. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    Published
    Feb. 18, 2016, 3:42 p.m.
    ...cloning a gene into an expression vector, you would want the start of the gene to end up just downstream ...verify that the insert ended up in the direction you want - usually by Sanger sequencing or further restriction...some DNA during the gel purification step, it is important to digest plenty of starting material. We recommend...be cut with both enzymes, and therefore it is important that the digest go until completion. The time ...running a gel for purification purposes it is important to have nice crisp bands and to have space to ... your favorite gel purification method, it is important to determine the concentration of recovered DNA...DNA in a standard ligation reaction. You ideally want a “recipient plasmid : insert ratio” of approximately...

  6. Building Global Connections with the International Mentorship Program USA-EUROPE

    Type
    Blog Post
    Published
    Nov. 6, 2015, 3:30 p.m.
    ...great improvements, not necessarily quantifiable, but always significant. Doubts, fears, insecurities… all...professionals has made possible a significant qualitative and quantitative expansion in the 2015-2016 academic...how to write cover letters, and has suggested important things to emphasize in order to be selected in...to Spain at one point, since I am not sure that I want to pursue the academic track.” How does an International...Scholars need strong proactive mentors who have relevant expertise and skills, positive disposition and... of Oviedo, the European University Miguel de Cervantes, the University of Navarra, and the University...pursue them, increased their network of contacts important for their career goals, and identified possible...

  7. Starter guide to induced pluripotent stem cells (iPSCs) part 1:  A renaissance in regenerative medicine

    Type
    Blog Post
    Published
    Oct. 30, 2018, 1:04 p.m.
    ...therapeutic purposes. Both types have their advantages and disadvantages. Here is a table listing some of them... system which combined the advantages of both while limiting disadvantages led to the creation of induced...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... mostly from abnormal and/or supernumerary preimplantation embryos that are produced during in vitro fertilization...these multipotent stem cells are located in small quantities in specific “stem cell niches” found in many ...during ESC isolation. Low Chances of post-transplantation rejection when used therapeutically High Low...similar functional utility, iPSCs bypass the transplantation compatibility issues and ethical concerns of...

  8. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

    Type
    Blog Post
    Published
    June 22, 2017, 2:30 p.m.
    ... and meaningful way it is important to choose the appropriate quantification methods and tools. It is ...living cells and whole organisms. You can take advantage of the many Fluorescent Proteins (FP) available...Once you've created your fusion protein, it's important to validate its function relative to the untagged...protein visualized with a fluorescently-tagged antibody. By employing “marker proteins” which are known... of a cell. This mapping can be done by using antibodies targeted to the marker proteins, however, in ... cell membrane permeabilized in order for the antibodies to reach their intracellular targets. Therefore... pipelines. Image analysis software produces quantifiable results and statistics that can be used to compare...

  9. Experimenting with New Careers while in Grad School

    Type
    Blog Post
    Published
    Feb. 25, 2016, 3:30 p.m.
    ...Medicine. It’s not always easy to figure out what you want to do after graduate school, at least not while ... into my PhD in Molecular Biology, I realized I wanted a career in science outreach: engaging the public...mentor and graduate office. I cannot stress how important they were to my success as a student. They didn...supportive of your career goals, find people who are. I wanted to do fun science activities with the public, so....  Try new things to further your career Take advantage of opportunities provided by your programs and...including workshops and networking events. I took advantage of as many of these resources as I could. Networking...front work was done for me. I just had to take advantage of it. Blog and tweet I started my blog, Born ...

  10. Drew Endy Introduces the Biobrick Public Agreement Plasmid Collection

    Type
    Blog Post
    Published
    Dec. 12, 2013, 3:35 p.m.
    ...gene systems, what do you want to make? So, tell us readers, what do you want to make?   References: Jerome...were so much fun and so incredibly useful that we wanted to make them free to use by everybody. (For more...YouTube primer on BIL gates.) Addgene: Why is it so important to you that these parts be made freely available...out of 15 are free. Now, the BioBricks Foundation wants to develop a language for programming life. We can...information into actionable signals. Let's say I wanted to detect every primary metabolite in a cell. The...language is put into practice? Endy: I think it's important to understand that these platforms, or pieces ...incredible is the bioeconomy is already pretty substantial. It's estimated that genetic engineering as applied...

  11. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    Published
    March 17, 2016, 2:30 p.m.
    ...sense, genome replicative intermediate (anti-minigenome). The anti-minigenome is used as a template by the...effectiveness of antiviral compounds which often target the viral polymerase. Technical tips It is important that ...viral proteins are provided in trans (see Other Important Components of EBOV Minigenome System). However...negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus minigenome... Firefly luciferase can readily be measured to quantify (indirectly) transcription performed by the viral...utilize the minigenome as a template. Thus, it is important to choose cell lines that are highly transfectable...plasmid transfected into cells are also extremely important. For example, particular ratios of plasmids encoding...

  12. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    Published
    June 27, 2018, 1:16 p.m.
    ... single domain antibody fragments typically engineered from heavy-chain only antibodies found in camelids...camelids. They possess many advantages over conventional antibodies: they are small (12-15 kDa), stable, ..., allowing for quantitative analysis using different detection methods. Since the anti-GFP nanobody is... RNA templates to DNA but suffer from several important shortcomings including low processivity (ie. the...MarathonRT, isolated from Eubacterium rectale, significantly outperforms other RTs in this regard and is ...still just as specific to their target as regular antibodies. These properties make nanobodies easier to transform...toolkit includes a set of plasmids each containing an anti-GFP nanobody fused with a different functional element...

  13. Science Career Options

    Type
    Blog Post
    Published
    Feb. 23, 2017, 1:30 p.m.
    ...becoming more reliant on technology, and good computer skills will give you an advantage. Strong writing...leaving academia, but don’t quite know what you do want to do or even what careers are available to scientists...your goal might not lead to a career you actually want, you can feel lost. Use this post to explore the...including: Livestock Breeder Scientific Illustrator Plant Breeder Zoological Worker Conservationist Documentary... Statistician Bioinformatician Science Writer Grant Writer Journal Editor Science Marketer Software... who currently in the position. Questions you’ll want to find answers to include: Will you be sitting...sitting at a desk all day or be on your feet constantly in a lab? Will regular travel be required or will ...

  14. Plasmids 101: Mammalian Vectors

    Type
    Blog Post
    Published
    March 25, 2014, 3:15 p.m.
    ... is possible that you may not want the plasmid depleted, but still want to use transient transfection ...Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T cells), allow ...mammalian cells is fairly straightforward and the resultant cells can either express the plasmid DNA transiently...reduces the rate of plasmid dilution but does not guarantee 100% transfection efficiency. Stable transfection...plasmid contains a cassette that will make cells resistant to a toxin), whereas negative selection would ...Before doing a transfection experiment, it is important to determine the proper concentration required...reagent). Cells should die within 3-5 days and resistant colonies appear in about 10-14 days depending ...

  15. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    Published
    Aug. 15, 2017, 1:24 p.m.
    ...PMID: 18193049.  9. Filipe-Santos, Orchidée, et al. "A dynamic map of antigen recognition by CD4 T cells...with the engineering of fluorescently labelled antibodies made it possible to track cells from one organ...harvesting lymph nodes at different times after antigen challenge, scientists were able to track where ...diverse tissue environments. These studies provide quantitative measurements of cellular motility, interactions...By specifically labeling T cells, B cells, and antigen presenting cells, scientists have been able to ...the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon microscopy enabled...scanning was and how fast T cells could jump from one antigen presenting cell to another. Indeed, we now know...

  16. Communicating Your Science With Help From ComSciCon

    Type
    Blog Post
    Published
    July 19, 2018, 1:12 p.m.
    ...if you cannot convince others that it is important and relevant to them.   When you think about it, virtually...this lead to the conclusions I want them to draw and the perspective I want them to form? Training in science...believe that communication is the single most important skill that scientists need to succeed in their...next interview or whether your receive the next grant you apply for. After all, the only value your work...high level to their team.  When you're writing a grant, this means convincing a committee that your research...your audience about the work you've done. If you want to maximize the impact of your communication, you...you will also have to explain why the work is important and how it should change how the members of your...

  17. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    Published
    Nov. 5, 2019, 1:28 p.m.
    ...the most recent advances and how some of these variants may be appropriate for your specific research ...nuclease for your experiments, there are three important factors to consider: site accessibility, specificity...potential for off-target cleavage should be an important consideration during any experiment. Sensitivity...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites (... be targeted (Hu et al., 2018).  These altered variants can allow for targeting of your genomic loci of...stringent PAM specificity. This can be especially important for base editing techniques, where Cas9 must be...many options! Comic by Maya Kostman. Another important consideration when choosing a Cas9 is balancing...

  18. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...In this blog post, I will go over some advantages, disadvantages, and examples of how scientists are using...commitment. Another advantage is that this process makes it easy to generate wild type and mutant constructs at... common, and for good reason. They offer many advantages over the traditional restriction enzyme cloning...example of using Gibson assembly, imagine that you want to insert your gene of interest into a vector with... have the tag already included in the vector you want to use. If you inserted these two pieces of DNA ...such as a hairpin or a stem loop (as might be anticipated to occur within a terminator sequence), since...specific for each fragment as well the fragment you want next to it, so this doesn’t allow for mix and match...

  19. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    Published
    Feb. 16, 2023, 7:05 p.m.
    ...mammalian cells: easy to use biological tools are abundant! In this post, we will review tried-and-true protein...well as explore some new innovations. Whether you want to turn things on or off, permanently or temporarily...requirements To pick the best tool for the job, it’s important to know what the job requirements are. Here are...temporary control (a few days) be enough, or do you want a permanent control mechanism? Where is the control...together. Turn it off Turn it off forever If you want to turn off protein expression, you are almost certainly...for genomic delivery and have many therapeutic advantages, such as tissue-specific targeting. Looking to...1 CRISPR activator and repressor mechanisms Want to transiently induce expression? Check out Addgene's...

  20. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    Published
    June 14, 2018, 11:44 a.m.
    ...years since its discovery, researchers have done significant work to improve the Adv genome’s utility in research... quickly due to immune response against AdV. Importantly, recombination-competent adenovirus (RCA) can...formation assay on A549 cells . Alternatively, antibody-based assays are commercially available for faster...more complex and the presence of helper virus contaminants have slowed the use of this system. New and ...detailed instructions on how to generate the final recombinant adenoviral plasmid construct, please see Addgene... references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into...2-3 weeks) - Here you’ll produce the primary recombinant adenoviral stock (rAdV-S). HEK293 cells are transfected...

  21. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    Published
    May 15, 2018, 12:43 p.m.
    ...degradation of caffeine (1,3,7-trimethylxanthine) and its conversion into xanthine. The majority of this operon...enzyme required for conversion of xanthine into guanine, and without xanthine supplied in the media, these ...desired due to its very low quantum efficiency. ShadowY has a lower quantum yield and higher absorption...material for the bioproduction of therapeutic methylxanthine drugs like theophylline. To facilitate future... glutathione S-transferase homolog from a Janthinobacterium sp. Moreover, a caffeine “addicted” E. coli... of this knockout strain can be rescued by the xanthine produced via complete demethylation of caffeine... assays. ShadowG is darker than other dark YFP mutants, it reduces the risk of spectral contamination ...

  22. Adeno Associated Virus (AAV) for Cell and Gene Therapy

    Type
    Blog Post
    Published
    Nov. 7, 2017, 1:59 p.m.
    ...seriously hinder gene therapy outcomes. Anti-AAV antibodies developed by the immune system – patients...To date, gene therapy has been used to treat 10 infants with this disease. To do so, their T-cells were...and the T-cells were transferred back into the infants. Almost all patients have achieved persistent immunological...immunogenic profile. AAV was first discovered as a contaminant of adenovirus preparations. It is an icosahedral...disease, and HIV. A notable landmark study showed significant clinical efficacy against a form of blindness... of LCA and mutations in RPE65 (n=3), showed significant improvements in vision over a period of 1 to ...infection. For example, prevalence of neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are ...

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

    Type
    Blog Post
    Published
    July 23, 2015, 2:30 p.m.
    ...to solve, we want to focus our research on worm locomotion, but our worms are constantly moving so we ...complexity using existing C. elegans mutants. For instance, we use a mutant known as CX51 that exhibits motor...real mutant worm. Like many academic labs, we have the capability to study many different mutants because...educational institution meaning that we can obtain known mutants at $7.00 a strain. Makerspaces like ours can also...model  so that we can use CRISPR/cas9 to build the mutants, analyse their motor behaviour, and ultimately ...month - with only occasional supplementation with grant funds, money is always tight. Of course, unlike ...monitoring homemade microfluidic chips using the fantastic optics and fluidics platform it contains. Biotech...

  24. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ...transform your resultant plasmid libraries into chemically competent E. coli and analyze transformants for appropriately...characterization, these proteins could function as surrogate antibodies for identifying and characterizing sugars and...Essentially, any method that results in the bulk of resultant fragments falling between 1 and 3 kb results in...culture, dilute and plate the complete library of transformants for single colonies (this usually requires 10...DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3) cells (these... protein production), then dilute and plate transformants for isolated colonies (150-200 per plate) across...then be exposed to a lysis buffer to release recombinant proteins directly onto the nitrocellulose for...

  25. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    Published
    April 4, 2019, 12:53 p.m.
    ... used to identify antibiotic resistance genes. Genes that are targets of antibiotics are often essential...for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First...customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9...downstream of the glmS gene in the recipient's genome. Antibiotic plates lacking DAP select against donor bacteria...The ICE integrates into the trnS-leu2 locus. Antibiotics select for recipient bacteria with an integrated...experiments where maintaining library diversity is important, while lower transfer rates are only suited for...researchers to study if that gene is a target of an antibiotic. For example, let’s take a look at the essential...

  26. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ...to try developing a variant with an enhanced response. Evolution of a new variant Maria and the team set...project, Maria’s screens of AsLOV2 variants showed some significant gains! After a few more rounds of ...mutagenesis and screening, the EGFP and mScarlet variants were showing no obvious signs of improved response...the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled to see that MagLOV’...: Fluorescence intensity and ΔF/F of MagLOV, a variant of AsLOV2 with mutations C450P, L496V, Q513K, G528K... Andrew and Maria expect that further-improved variants of MagLOV will be possible with other engineering...other researchers from a range of fields, like quantum chemistry and protein biophysics, as well as inventors...

  27. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    Published
    April 19, 2016, 2:30 p.m.
    ...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent ..., and behavior of a POI. As an investigator, you want a fusion tag to be inert, to not produce artifacts...making FPs monomeric, yet many investigators remain ignorant of FP dimerization. Equally problematic, several...its molecular affinity, termed the dissociation constant or Kd, and its concentration. The smaller the ...volume. In many biological research situations, the relevant volume is that of the cell. For the physical behaviors... Fusion of an FP to a transmembrane protein significantly increases the effective concentration of the...biosensors were made with CFP and YFP, cyan and yellow variants of EGFP equally capable of dimerizing as EGFP....

  28. Introducing Addgene's New Search - Find Plasmids More Easily For Your Research!

    Type
    Blog Post
    Published
    Dec. 3, 2019, 11:38 a.m.
    ...example we’re going to be looking for recombinant monoclonal antibody plasmids from the James Trimmer Lab...better. Here, I wanted to find a specific plasmid from the NeuroMab collection for an antibody against PARIS... terms used, so you will only be shown filters relevant to your current search. We’ll take a closer look...look at filters in a moment. First, I want to highlight the convenient ways our new search tool lets you...distributing 94 plasmids expressing validated antibodies for neuroscience research (see the NeuroMab Collection...plasmid that would be useful in an experiment, and want to see if someone has already created and deposited...and filters change as we now only show options relevant to your current search terms and potential results...

  29. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ...either transcript variant led to robust and specific upregulation of the targeted variants without inducing...activation? To summarize, CRISPRa uses a nuclease-dead variant of Cas9 (dCas9) and engineered combinations of ...Figure 1a for the transgenic construct). More importantly, the expression of this construct is Cre-dependent...used their optimized vectors to highlight a key advantage of CRISPRa over traditional cDNA overexpression... gene isoform individually. To highlight this advantage, they selected the immediate-early gene Bdnf, ...inducing expression of the non-targeted variant. Moreover, isoform-specific expression of Bdnf resulted in... between the Bdnf I and Bdnf IV overexpression variants were other immediate-early genes associated with...

  30. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    Published
    April 2, 2019, 1:44 p.m.
    ...genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important for...protein Groucho, a transcriptional co-repressor important in development. Groucho family proteins are found... in neural development, sex determination, and anterior-posterior segmentation (Jennings and Ish-Horowicz...Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest...video game character but rather to an extremely important gene in animal development. To understand where...name but some of our Addgenies had an idea: sqh mutant fly ovaries had “squiggly” actin fibers that resemble...spermatocytes to enter meiosis. Testis of cookie monster mutant flies are filled with early mitotic cells as well...
Showing: 211 - 240 of 884 results