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We narrowed to 1,018 results for: PLE

Showing: 551 - 600 of 1018 results
  1. Troubleshooting Your Plasmid Cloning Experiment

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    Blog Post
    ...Assembly, Golden Gate etc.), and try to find the simplest way to do it (i.e. avoid assembling too many fragments...methylation like XbaI, ClaI, etc. If you are doing multiple digestion be sure that the buffers and temperature...Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to assemble the  fragments...molar ratio. Assembly of the fragments There are multiple ways you can assemble the different parts of a...plasmid. Many vectors contain sequences that are complementary to standard sequencing primers. If not, design...
  2. Transferable Skills Guide: Problem Solving

    Type
    Blog Post
    ...ill-defined. In this article, I try to clarify what people mean when they ask about “problem solving skills...problem.  Step Three: Decide on a strategy and implement it.  Step Four: Assess how effective your strategy...the cause. To test the most likely cause, you implement an experiment designed to fix the problem. Then...modifications, and ultimately decided on the changes to implement. As with any skill, there will always be room ...Know your strengths! Research experience presents ample opportunities to practice problem solving skills...
  3. The time and cost required to make a plasmid

    Type
    Blog Post
    ...outlined in Table 1.   Of course, this is for the simple insertion of a PCR product into a vector. The time...calculate, in both our 2018 and 2023 poll, most people said it took them more than 22 hours, so at least...published in. That’s also a great way to get to know people doing interesting and relevant work in your field...That way, you can run one PCR, one gel, etc. for multiple plasmids at once. Over time, this can save you... Multiple Authors...
  4. CRISPR 101: Cas9 vs. The Other Cas(s)

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    Blog Post
    ...is guided by an RNA molecule, the gRNA, to a complementary site in the genome where it will proceed to ...degradation. Cas3 DNA digestion can proceed up to multiple kilobases, but only in one direction. Cas3 is ...that Cas14 also indiscriminately cleaves non-complementary ssDNA when engaged by its target sequence. Why...used in lieu of siRNA or shRNA for efficient, multiplexable RNA knockdown. Cas13 has also stirred up interest... the other Cas family members make for useful implements in the scientist toolbox. If your genome engineering...
  5. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

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    Blog Post
    ...ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate while expressing...system contains ccdB. A successful insertion will completely replace ccdB with the investigator’s insert of...interest.   CcdB resistant E. coli strains complete the system But ccdB alone isn’t the only key to...most appropriate method for your own experiments. Please check out Addgene’s Choosing a Molecular Cloning...protein involves poisoning of DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed...
  6. Gendered Innovations: Why Does Sex of the Cell Matter?

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    Blog Post
    ...conduct research that fails less and helps more people, we need to take sex into account. Gendered Innovations...s Health Initiative Hormone Therapy Trial, for example, found that the study saved lives. Reduced use ...analysis and 2) provides case studies with concrete examples of how gender analysis leads to discovery and ...experiments—which can lead to failed research. For example, an international research team from Norway and...studies that draw from a body of research that is incomplete (because sex was not analyzed) may harm humans...
  7. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

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    Blog Post
    ... the xanthine produced via complete demethylation of caffeine. For example, the Barrick lab was able to...ever like to write about a recent plasmid deposit please sign up here.     Listen to the Hot Plasmids episode...living biosensor for measuring caffeine content in samples. The Barrick lab and the UT Austin undergraduate... to grow this strain in minimal media supplemented with various caffeinated beverages, but not with caffeine-free...
  8. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

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    Blog Post
    ...CRISPR is a simple and versatile tool for genome engineering, but its utility is dependent on its ability...tiny DNA cutting ninjas that deliver CRISPR to multiple types of targets: primary cells, embryos, and ...helps form DNA and retroviral-particle or VLPs complexes. The team found that "all-in-one" Nanoblades facilitated...their CRISPR cargo into the zygote. As a proof-of-principle, the team used Nanoblades to disrupt the tyrosinase...no detectable editing. Additionally, off-target spleen editing was not detected and Nanoblade injection...
  9. Getting Started with Behavioral Neuroscience: Standardizing Experiments and Using Viral Vectors

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    Blog Post
    ...coordination to more complex situations such as learning and memory or emotional states, for example anxiety or ...and cause variability in the testing group. For example, if an experimenter picks up a mouse from the home...kHz, produced from fluorescent light bulbs, for example) that are outside the humans’ audible spectrum.... have preferences for specific cell types. For example, AAV1 and AAV9 serotypes have strong neuronal tropism...
  10. Open Resources and Plasmid Tools For Studying C. elegans

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    Blog Post
    ...all essential embryonic genes are individually depleted via RNA interference (RNAi). Scientists monitor...AID system discovered in plants to conditionally deplete proteins of interest in C. elegans (Zhang et al...Schwartz and Jorgensen, 2018).  These are just some examples of worm resources and tools openly shared by the...Hammarlund, Marc, et al. "The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System... system enables versatile conditional protein depletion in C. elegans." Development 142.24 (2015): 4374...
  11. Hot Plasmids - October 2020

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    Blog Post
    ...ever like to write about a recent plasmid deposit please sign up here.  Here's what you'll find in this ...expanding our ability to study and understand the complex interworking of the 86 billion neurons in the human...Using genetically encoded fluorescent proteins coupled with Fibronectin intrabodies generated with mRNA...speed across these different cell types. In one example, H2B-FT was expressed in murine B cells (BaF3),... the plasmids at Addgene The Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA) ...
  12. Design Tips for Prime Editing

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    Blog Post
    ...extended to include a primer binding site (PBS) complementary to the genome and a reverse transcriptase (RT... the experiment. Now, onto the tips! General principles pegRNA design If you’re just starting out with... nicking the newly synthesized strand before heteroduplex resolution, which can lead to indels (Anzalone...edited strand as a template during DNA repair. Test multiple nick sites, starting with sites about 50 bp upstream...Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell, 156(5), 935...
  13. Adeno Associated Virus (AAV) for Cell and Gene Therapy

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    Blog Post
    ... the mutation or replacing the gene. A notable example is the treatment of Severe Combined Immune Deficiency...normally functioning T cell repertoire (1). In this example, it was possible to transduce the T-cells outside...based delivery techniques may hold the key. In the simplest form, translating gene therapies from conceptual...already have ways to prevent AAV infection. For example, prevalence of neutralizing antibodies against ...: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates gene targeting and enhances endonuclease...
  14. 6 Tips for Grant Writing

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    Blog Post
    ...bit of advice I can suggest, is to give yourself plenty of time when writing a fellowship proposal. Yes... you learn new techniques and interact with new people. If you are looking to move to a different university...there is an increasing realization that, if we want people to keep contributing to science, it would be helpful...audience. My lay paragraph typically includes a couple of sentences of introduction to my area, a line...great information on ELISA troubleshooting and multiplex assays.   Additional Resources on the Addgene ...
  15. Savvy Advocates Needed to Navigate a Scientific Enterprise in Flux

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    Blog Post
    ...challenges and opportunities for us all.  For example, implementation and compliance for the FLSA changes, including...turn. The first is the passing and upcoming implementation of the Federal Labor Standards Act (FLSA) which...remain mixed in their opinions on the subject – for example, Yale graduate students filed for union elections...challenging, but will be aided by the development and implementation of new strategies designed to cope with current...
  16. Tips for Titering Your Lentiviral Preps

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    Blog Post
    ...titers measure the amount of viral particles in a sample and are usually based on the presence of a viral...however, is that a p24 ELISA measures all p24 in the sample regardless of whether or not it is incorporated...measures infectious viral particles. One of the si mplest methods of measuring functional titer uses FACS...integrated into its genome and a cell that has had multiple integration events. In addition, promoter choice... cell’s genome. This approach can discriminate multiple integration events, reducing the possibility of...
  17. Transferable Skills Guide: Managing a Team

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    Blog Post
    ...I knew when I first became a manager. Know your people Each person is unique and should be managed accordingly...accordingly.  I wish I knew that when I was “managing” a couple of undergrads back in grad school. Both were hard-working...Myers-Briggs test:  my undergrads were different people! More specifically, they had different learning... saying “I’ve been doing some of X’s work for a couple of weeks now”. I had no idea this was happening... and maybe even the slight awkwardness can spur people to speak up. Once aware of the problem, I was able...
  18. The Scientific Conference Poster Session: Tips for Success

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    Blog Post
    ...criticism, and ask thought-provoking questions. For example, if you’re a virologist presenting at the American...like to say to help build your presentation. For example, when thinking about discussing a figure, jot down...practice, practice: Rehearse your presentation with people inside and outside your field. Your colleagues,...brainstorm potential questions you may receive. People often ask about the relationship of your work to...guilty of this, and have many times answered a completely different question than the intended one. Take...
  19. 3D Printing Meets CRISPR Cas9

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    Blog Post
    ...realized how perfectly flat the complex is on this surface”….where the complex is docked on the membrane of...like “Oh,…I didn’t quite appreciate that….” For example, when Tom Steitz was presented with a physical ... close to its reported cleavage site on the complementary DNA strand. And this is why we love working ...double-stranded DNA, recognize a PAM sequence, allow the complementary base pairing between the guide RNA and the target...
  20. Fluorescent Proteins 101: Fluorescent Protein Timers

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    Blog Post
    ...and look at them under a microscope, fractionate samples to isolate particular organelles and their contents...fluorescent proteins have usurped old methods or complemented them in ways that make them much easier. A special...localized to a specific region of the cell. For example, if the ratio of red/green fluorescence falls within...experimental setup less cumbersome. 3. Portability across multiple systems Terskikh et al. showed that their versatile...in gene expression. In a developing embryo, for example, a researcher could determine whether or not newly...
  21. Production of Virus in Insect Versus Mammalian Cells

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    Blog Post
    ...specific type of virus you are trying to make. For example, bacteriophages will need to be produced in bacteria...cells. However, some viruses can be produced in multiple different cell types, including plants, mammals...supernatant is typically collected over the course of a couple days to increase yield. The collection process ...preps, lentiviral titration requires an additional couple of days, as it requires transduction of more cells...titrated following purification using qPCR or digital droplet PCR. Insect cells Insect cells are a less common...
  22. Hot Plasmids: Winter 2025

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    Blog Post
    ... ONE-GO biosensors kit By Mike Lacy G protein-coupled receptors (GPCRs) are critical components of many...detection of activation triggered by endogenous GPCR. Example applications include large-scale pharmacological... your research! If they do (or if they don't!), please consider sharing your experience with other researchers...recombinant antibody in human postmortem brain samples. Results show positive staining in tissues from... Multiple Authors...
  23. Anatomy of a Plasmid Page at Addgene

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    Blog Post
    ... plasmid page is nested in. You can see in the example above that the bread crumbs trail starts in the...  These sequences are provided in FASTA format (example) and can be analyzed using Addgene’s Analyze Sequence...Price of course! The price of some items vary, for example, plasmid kits are avaliable for less than than ...which experimental systems it was validated in.   Please keep in mind that all of the information on our...ever have any questions about our plasmid pages, please don't hesitate to contact us at [email protected]...
  24. AAVs CREATed for Gene Delivery to the CNS and PNS

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    Blog Post
    ...increase transduction efficiency, none allow for simple and efficient transduction of both the CNS and ... not lost in a jumble of fluorescent neurons. A simple but flawed approach to multi-color labeling is ...lots of individual cells will be transduced with multiple viral particles and labeled a variety of colors...the CNS (figure 5 in Chan et al) and the PNS (supplemental figure 8 of Chan et al). These new AAVs can ... transgenic mouse required (see figure 5 and supplemental figure 8 in Chan et al).  References 1. Chan...
  25. Viral Vectors 101: The Retroviral Lifecycle

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    Blog Post
    ...vectors. Lentiviruses are sometimes referred to as “complex retroviruses ” (Cullen, 1992) because of their ... we will mostly use the gamma-retroviruses as a simple spokesmodel for the whole family; however, you ...beginning to realize that the situation is already complex enough that a deep understanding of retroviral ...polymerase gene may seem like a mystery. But it’s simple. The RT protein is made by a separate set of instructions...Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375...
  26. CRISPR Antimicrobials

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    Blog Post
    ...using multiple approaches. Major restaurant chains including Chipotle and Panera Bread have pledged to eliminate...unprepared. Each year in the United States, two million people will be infected by antibiotic resistant bacteria...antimicrobials, and they suggest that targeting multiple resistance/virulence factors may further reduce...methods. Phage cocktails could be used to target multiple types of pathogenic or drug-resistant bacteria...
  27. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

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    Blog Post
    ...with the viral polymerase, which is formed by a complex of two proteins: the large (L) catalytic protein...-stranded RNA minigenome. The viral polymerase complex then recognizes the encapsidated minigenome as ...efficiently reconstitute a functional viral polymerase complex and utilize the minigenome as a template. Thus,...transfected into cells are also extremely important. For example, particular ratios of plasmids encoding NP and ...mRNAs and minigenome. This can be accomplished in multiple ways: transfecting with an additional plasmid ...
  28. Plasmids 101: Protein tags

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    Blog Post
    ...blots, IP, and affinity purification. Arguably the simplest affinity tag is the polyhistidine (His) tag. Small...Unlike other epitope tags – which when doubled or tripled increase the tag size quickly – modifying the length...localizes your protein to the nucleus? Or you want multiple rounds of purification to get your protein as ... a few time-tested tags and fusion proteins as examples. The tables list more common tags than are described...Biotechnology Journal 2012 , (7): 620-634. Full Text A complete list of free handbooks for protein purification...
  29. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

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    Blog Post
    ...genes in Strongylocentrotus purpuratus, the spiny purple sea urchin. It’s not a standard model organism,...editing in squid is possible nonetheless - the first example was of a pigmentation gene.   Genus Microtus (Zoe...Donaldson) Voles have fascinating social behavior. For example, two similar species have very different social...babble as baby birds, and have dialects just like people. This is actually rare in the animal kingdom. Gasterosteus... most grown crop in the world so it feeds a lot people. This lab is trying to improve homologous recombination...
  30. How to Write a Scientific Review Article

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    Blog Post
    ...citations so that people can go back and read the original reference for the data. For example, we summarized...sense of the themes in the field and to learn what people cared about. I also used reviews to get a list ...original paper if you wanted to learn more. Another example: we made a schematic of all the proteins in the...paraphrase what I have heard many other creative people say about writing: you don’t know what it’s going...
  31. Rett Syndrome: A History of Research and Therapeutic Outlooks

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    Blog Post
    ...). Neurology 92 (15 Supplement) . http://n.neurology.org/content/92/15_Supplement/S51.002.abstract Sinnamon...Cre not only halted the neurological damage but completely reversed it, regardless of age or symptom severity... Rett Syndrome MECP2 follows the “Goldilocks” principle, in which too much of the MECP2 protein is as ...editing can repair a gene in a living animal across multiple cell types using a mouse model for a human neurological...
  32. To Codon Optimize or Not: That is the Question

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    Blog Post
    ...the anticodon,  a three nucleotide sequence complementary to the mRNA sequence. The 3’ end of  the tRNA... the 20 amino acids and three stop codons. (B) Example of codon usage among different organisms. There...prokaryotic systems or yeast (Lanza et al., 2014). For example, it may be worth codon optimizing a human gene ...optimized for expression in mammalian cells. For example, Henry Lester’s lab optimized the C. elegans GluCl...wobble base-pairing (a tRNA that can recognize multiple synonymous codons) may actually be critical for...
  33. Twenty Years of Sharing: Expanding Accessibility

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    Blog Post
    ...easy to share plasmids, both for people depositing plasmids and for people requesting them. Prior to this... and remove these logistical barriers, allowing people to access a wide catalog of plasmids from all over... solve an issue specific to that country. ‌For example, when we began offering temperature-sensitive materials...inclusive design in our digital content. We have implemented proactive measures to find potential barriers...
  34. Hot Plasmids - November 2023

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    Blog Post
    ....), while PE6c and PE6d were most efficient at complex edits, such as long or highly structured templates...different viral families and can be used to target multiple delivery systems, such as lentivirus and MMLV ...tet-responsive element (TRE) or the cFos promoter. For example, neurons treated with a phorbol ester activator...recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain. eLife, 8, e43322. doi... Multiple Authors...
  35. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

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    Blog Post
    ...four-base overhangs flanking each genetic part. (A) Example Level 0 assembly. BpiI recognition sites and four-base... Level 0 acceptor (bottom) for CDS1 parts. (B) Example Level 1 assembly. The choice between different ...important that these stay frozen at -80 °C, since multiple rounds of freezing and thawing will affect bacterial...plasmids with undesired ligations, so always screen multiple colonies after each transformation. This could... MoClo user, but beginners or those performing complex assemblies should screen a minimum of 8–10 colonies...
  36. Five Popular Model Organisms, Part 2

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    ...manipulated for use in molecular biology studies. For example the E. coli biotin ligase, BirA, has been used ...corresponds with human diseases and conditions. For example, the rat is frequently used to study human cardiovascular...abilities. The axolotl has the ability to regenerate complex structures such as limbs, the spinal cord, and ...them an ideal model organism; they are relatively simple to maintain and breed and they have large, easily... and microinjections. Injections of mRNAs, for example, can be analyzed to understand human disease alleles...
  37. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

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    Blog Post
    ...cells will be co-transduced with multiple copies of both vectors. Examples of use: Halbert et al., 2002...then following transduction, the partial genomes complement each other to restore the full-expression cassette...outperform single oversized AAVs (Hirsch et al, 2013). Examples of use: Lai et al., 2005 used a trans-splicing... the accumulation of such truncated proteins. Examples of use:  Ghosh et al., 2008 used a hybrid split...
  38. CRISPR 101: Validating Your Genome Edit

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    Blog Post
    ... words, any insertion or deletion that isn’t a multiple of three will do. A popular screening method for...trace file generated in Sanger sequencing and a simple algorithm to quantify editing efficiency by insertion...have to screen to identify a knockout clone. For example, if your cell line is diploid (2N) and your out...to compare the sequencing reads from your edited sample to an untreated population. Software such as CRISPResso... Multiple Authors...
  39. Using Phosphoserine to Study Protein Phosphorylation

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    Blog Post
    ...protein phosphorylation in an array of model systems coupled with advances in mass spectrometry suggest that...recognizes a specific ‘open’ codon and utilizes this triplet to incorporate phosphoserine A Sep-tRNA synthetase... purified from the system, researchers can run samples on SDS-PAGE gels containing Phos-Tag, a small molecule...substitution. Alternatively, you can express the completely wild type protein from the same plasmid.  Upon...reference and protocols for use of this system, please reference ‘Rinehart Lab reagents for improved expression...
  40. Is this the right place for me? 8 tactics for choosing a lab

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    ...emotions and assumptions. I’m not just talking about simple things like “Which route should I take to get to... give you a fuller picture. Consider talking to people in the lab across the hall or in the same department...choosing a training lab, I strongly suggest you complete a work rotation in the lab if at all possible.... lab members alone during the interview period. People seem to be hiding their work and are nervous about...successful. Collaborative Competitive Papers have multiple authors One author plus PI on most papers Productive...
  41. CRISPR 101: Off-Target Effects

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    ...goal of isolating single cell clones to use for multiple downstream experiments. You only successfully ...specific list of the candidate off-target sites. For example, if there are 10 putative off-target locations ...most commonly used Cas, spCas9, has gone through multiple rounds of optimization which have led to more ...confounding your results should be minimal. For example, if your goal is to generate a knock-out cell line...are performing CRISPR library screens, the same principle holds true - the higher the number of distinct...
  42. 8 Great Depositor Research Stories

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    ...us about a new tool for efficient multiplex base editing and multiplex prime editing (requiring minimal...editing tool for large inserts.  DAP arrays for multiplex gene editing: Guest blogger Qichen Yuan tells ...a research story you want to share on our blog, please reach out to [email protected] - we'd love to hear...
  43. Our New Antibody Mascot is....

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    Blog Post
    ...journey is at an end! Their name is...(drum roll, please!)... Abi! Thank you so much to everyone who submitted... name. So we reached out to you and received a plethora of options, ranging from Affie to Yncredibody....Which name best fits our new immunoglobulin friend? Please help us decide by voting for your favorite on this...Original Blugene and Aavery would like your attention, please, as they introduce their new friend…  Fig...
  44. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

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    Blog Post
    ...antibodies, for example through the use of less toxic adjuvants; 2) generating complex antibody chips ...cells are fused with activated B cells from the spleen of mice injected with the antigen of interest. ... assessment of many protein targets in a single sample; 3) producing recombinant, partially humanized ...
  45. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

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    ...Bernstein and Boyden, 2011; Mei and Zhang, 2012). For example, blue light activates channelrhodopsins that, in...Abdallah et al., 2018; Deubner et al., 2019). For example, Taylor and colleagues used rAAV FLEx-based vectors... Han, 2016). Not so long ago, however, it was completely unclear which subpopulation of DA neurons could.... "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit ...Walther Stoeckenius. "Rhodopsin-like protein from the purple membrane of Halobacterium halobium." Nature new...
  46. Antibodies 101: Antibody Engineering and Directed Evolution

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    Blog Post
    ...methods. (A) Basic structure of an antibody. (B) Examples of engineered antibodies, including chimerization...achieve whatever goal you’re aiming for. When you complete each cycle, your best-performing mutant is usually...or predicted structure of your antibody-antigen complex as a template. This structural framework then guides...specific functionality you’re looking for. For example, if you were aiming to increase an antibody’s binding...conditions. These methods are especially powerful when coupled with iterative rounds of selection and mutagenesis...
  47. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the ...dsDNA was short enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects the...created by Exo and promotes its annealing to a complementary ssDNA target in the cell.  Only Beta expression...region of interest and design an oligo that is complementary to the lagging strand. In practice, it’s easier...system once recombinant clones are generated.  A simple way to do this is to express lambda red genes from...
  48. How Do I Place an Addgene Order Using a PO Number?

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    Blog Post
    ...Addgene, we try to make the process as smooth and simple as possible. We are the Customer Support Specialists... Placing an order on Addgene’s website is quite simple, even if you’re using a PO number. Follow these...generated through your organization in this field. Please also enter the billing address for your organization...time, your organization will have net 30 days to complete payment for your order.  Still have questions?...
  49. A Conference By Postdocs For Postdocs: Future of Research

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    Blog Post
    ...competition is fierce. However, I believe there are plenty of great jobs out there for science PhDs. The problem...organizers are building valuable skills and meeting new people by working together on the event planning and execution... execution. I am seeing many similar examples of science trainees taking a proactive role in their non-bench... Consulting. I know of many schools that have completely student/postdoc-driven biotechnology clubs such...
  50. Microbes: Look, Listen, and Tell

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    Blog Post
    ...visualization in vivo using ultrasound (for an example see the figure below). In addition to low costs...benefits of this technique. It is even possible to multiplex ultrasound dyes because these dyes “collapse” ...Approaches to Presenting the Microbial World’ showcased complex connections and ideas in various visualized approaches...gifs created by the Amoeba Sisters. Here’s one example of how they “bridge the jargon gap using gifs”:...
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