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We narrowed to 919 results for: IRE;

Showing: 201 - 240 of 919 results

  1. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    ...component required (e.g. dsODN), doesn't require high read counts (fast multiplexing) Requires large cell...genome for all DSBs, doesn't require high read counts, fast multiplexing Requires delivery of dsODN (potentially...length of the gRNA given the stricter sequence requirement at the terminal 3’ PAM site (Fu et al. 2013; ...by sequencing (GUIDE-seq) (Tsai et al. 2015) and direct in situ breaks labeling, enrichment on streptavidin...BLESS detect double-stranded breaks and do not require high sequencing read counts making them fast and...contains both the rare off target event and the desired edit is low. Therefore, unbiased sequencing may...selected. Conversely, translational research may require the rigor of both forms of off-target analysis ...

  2. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    ...editing systems; first, RNA editing doesn't require homology-directed repair (HDR) machinery, and could thus...in non-dividing cells. Cas13 enzymes also don’t require a PAM sequence at the target locus, making them... sequence, but orthologs like LwaCas13a do not require a specific PFS. Cas13 enzymes do not contain the...domains responsible for DNA cleavage, so they cannot directly edit the genome. A Cas13-based RNA editing system...enzyme for further studies. PspCas13b does not require a PFS, and it is sensitive to mismatches in target...the lowest amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced...activity. They then performed sixteen rounds of directed evolution across the full ADAR2DD to further optimize...

  3. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    ...Klompe et al., 2019; Strecker et al., 2019), which direct RNA-guided transposition. By natively combining...I-F CRISPR-Cas system. The four major components required for RNA-guided DNA integration are (Fig. 1A, top...transposition in type I-F INTEGRATE. The requirement for multiple Cas proteins to form the Cascade...targeting (Halpin-Healy et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by...instead of three Cas proteins (Fig. 1A, bottom), and require only two transposase proteins (TnsB and C). While...effector plasmid (pEffector) expresses all seven required genes and the CRISPR RNA under the same promoter...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...

  4. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...fluorescent donor to your protein is to hardwire the molecule directly into the protein’s peptide sequence...(the donor) is excited, it can transfer energy directly via resonance to the second fluorophore (the acceptor...translation (Ermolenko et al., 2007). All that is required is that the proteins/nucleic acids/lipids of interest...delivered to the right place? FRET experiments require targeting the donor and acceptor fluorophores to...experimental design is that the targeting specificity requirements are different for the donor and acceptor fluorophores...codon (often amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural...overhaul of the cell’s native tRNA synthetases is required and thus far, this method has proven most effective...

  5. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    ... ori and requires the host proteins DnaA and IHF to initiate replication. But pSC101 requires a third ...RepA binds to directly repeated sequences called iterons located near the ori and directly interacts with...said to be under relaxed control, as they only require the host's replication machinery for elongation...have enough nutrients available to complete the entire round of replication. Initiation is the primary...versions, but binding of the low-affinity versions is required to start the replication process. When enough ...creates two replication forks that move in opposite directions away from the ori. In the elongation phase, a...on host-encoded proteins, it can be regulated indirectly via the same mechanisms that affect DnaA and ...

  6. Management for Scientists: Giving Feedback

    Type
    Blog Post
    ...is necessary to be direct and clear. There is place in human discourse for indirect language, but it is...Giving timely and useful feedback is an absolutely required aspect of being a successful manager. Great bosses...over, what would you do differently? Beware of indirect language You must be honest about performance ... should be aware that they are at risk of being fired and should be looking for a new position. One of... mistakes I see is managers who hint at or use indirect language to talk about real performance issues...issues. Watch this little video to learn more about indirect language from Steven Pinker. As a manager you ...Scientists. Subscribe to the Addgene Blog to follow the entire series as it is published. For more reading try...

  7. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ... for a site-directed mutagenesis PCR that uses mutagenic primers to introduce all desired codon changes...generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure...phage-derived uracil labeled template, this method requires further DNA shuffling to combine pairs of mutations...of your liking. This method is similar to site-directed mutagenesis (SDM) PCR, but with a twist since ...the plasmid. Again, if they are in the opposite direction, both strands of the plasmid will be nicked during...of your plasmid. If the sites are in opposite directions, you just need to use a plasmid with BbvCI restriction... the gene or use custom gene synthesis or site directed mutagenesis to remove the BbvCI site(s). Overall...

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

    Type
    Blog Post
    ...practices, their opinions towards ELNs, and their desired functionalities i.e. main priorities of different..., and the fact that long-term use is likely to require on-going maintenance and support.” Besides that...it as very important. One comment reflected the desire for a flexible generic solution, rather than an...switching from paper to an electronic lab notebook requires time and knowledge. In a recent article, we translated...reporting on a project – printing your data out can be required every once in a while, so it is good to make sure...your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering this... that take any ownership over your data or have direct access to it. Your data should be kept private ...

  9. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...but this approach would require robust stem cell editing to maintain the desired phenotype. Precision genome...skipping. Addgene depositor Charles Gersbach used paired zinc finger nucleases to remove exon 51 in DMD ...For such a therapy to be successful, multiple requirements must be met. First, CRISPR would need to be ...especially well suited for genome editing. The homology-directed repair (HDR) pathway is downregulated in mature...is maintained for at least six months. Future directions and obstacles to clinical CRISPR editing Given...NHEJ-mediated editing, but most other diseases will require HDR-mediated precision editing tailored to smaller...:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya...

  10. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    ...the requirement for digestive motility to not be disturbed, the brain fibers could not be directly repurposed...constantly influenced by signals it receives from the entire body, making it difficult to probe due to the lack...monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes of interest, and stimulate...blocks of polymers are heated up and pulled to a desired thickness. The fibers are drawn with microelectronics...the fiber horizon? The brain interfaces with the entire body, so the obvious next applications are to expand.... P. (2020). Modular Optoelectronic System for Wireless, Programmable Neuromodulation During Free Behavior... Multifunctional microelectronic fibers enable wireless modulation of gut and brain neural circuits. Nature...

  11. Management for Scientists: Delegating is Key

    Type
    Blog Post
    ...appropriate plasmids, and code the entire page. That would be entirely too much work for one person, it ...Clearly articulate the desired outcomes. Begin with the end in mind. Specify desired results. Are you delegating...and are experts in each of the individual tasks required to develop the web page. The team manager must... expert) the end result is high quality and the entire process is completed more efficiently. Listen to...criteria for delegating tasks appropriately: Are the desired goals and outcomes clear? If you don’t have clear...results. What does success look like? Agree on desired outcomes. Concern yourself with what is accomplished...

  12. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    ...CyOFP1-NLuc-CyOFP1; Antares). Direct injection of plasmids for Antares or firefly luciferase into mouse livers... like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase...brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase. This is important...for how sensitive you can make a reaction. NLuc requires 100-fold less protein to produce the same amount... wavelengths (Fluc emits at 610 nm) and do not require external light sources. Unfortunately, detecting...detecting traditional luciferases’ dim light emissions requires long acquisition times that may be impractical...changes in intracellular calcium and is useful for direct imaging of events in cells and brain explants. ...

  13. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    ...absolute measure of target abundance, does not require standard curves, and can theoretically detect a...what kind of assays can be used to sniff out your desired mutations. The assays you will use aren’t that ...either a donor sequence integrated through homology-directed repair (HDR) (1), or indel mutations resulting...non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation of NHEJ to generate... and a NHEJ/ “drop-off” probe designed to bind directly at the predicted double strand break site (Figure...quantification. Comparatively, mismatch assays require between 200 and 500 ng of purified PCR product.... those with the bi-allelic mutations typically desired when screening clones for complete functional knockout...

  14. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    ...are measured. While the entire genome can be knocked out with CRISPR, the entirety of the small molecule... another way, only cells or organisms with the desired phenotype will survive and be assayed at the end...phenotype in the surviving population. Selections do require there to be a culture condition that can apply ... have to be addressed with screens. Selections require there to be a culture condition which eliminates...then assay cells which survive as these must have acquired a favorable mutation. But if the cellular pathway... with cellular redundancies, a screen would be required to answer your experimental question. Now that....1246981. Leemhuis, H., Kelly, R.M., Dijkhuizen, L. Directed evolution of enzymes: Library screening strategies...

  15. Plasmids 101: Yeast Vectors

    Type
    Blog Post
    ...Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenance within an E. coli cell... across many different cell types and each one requires different elements for vector propagation. This...cerevisiae; however, we’ve also noted some features required for the replication of fission yeast, Schizosaccharomyces...These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination...for use in S. pombe, on the other hand, do not require a well defined ORI. Instead, the size and A-T content...organism to synthesize a particular organic compound required for its growth. Many auxotrophic strains of yeast...they have incorporated the plasmid carrying the required gene.  The table below lists some of the most...

  16. Visualizing Protein Turnover In Situ

    Type
    Blog Post
    ... antibody was directed toward a protein of interest but the second antibody was directed toward AHA. Now...these experiments are problematic because they require radiolabeled amino acids. The use of radioactive...radioactive materials is highly regulated, requires designated work areas and come with health risks. Importantly...Erin M Schuman, a former Caltech researcher, now Director at the Max Planck Institute for Brain Research... In the standard PLA assay, primary antibodies directed towards two endogenous proteins of interests, ...Central PMCID: PMC300121. 3. Söderberg, Ola, et al. "Direct observation of individual endogenous protein complexes... PubMed PMID: 17072308.  4. Dieck, Tom, et al. "Direct visualization of newly synthesized target proteins...

  17. 5 Steps to Writing Grant Proposals for Grad Students

    Type
    Blog Post
    ...graduate students and postdocs will be hired, direct and indirect costs, etc. It may be helpful to ask ...Research at universities typically requires funding from a variety of government and academic institutions...skills, Elucidates the big picture and future directions of the project, Outlines a detailed scope of...! Additionally, keep in mind that universities require application submission in-house approximately 2... RFA contains all of the important information required for a successful grant application. It typically...avoidance of ‘to be’ verbs creates strong, well-directed meaning and delivers strength to the sentence....

  18. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...Nammour, J., Maiz, D., Dujardin, S., Ramirez, S. H., Hudry, E., & Maguire, C. A. (2019). Selection of an Efficient...(2021). Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery...off-target expression, it may be better to choose a direct delivery method over systemic delivery. Otherwise... packaging Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict...expression to specific tissues and cell types. If desired, spatiotemporal control of transgene expression...and astrocytes located in the CNS, however, it requires high doses. Requests have shifted toward the more...effectively transduce astrocytes or oligodendrocytes when paired with appropriate cell type-specific promoters or...

  19. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    ...E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  a guide RNA...accessible. Image from Gaudelli et al., 2020. Directed evolution identifies adenine base editors with...editing in cell lines Nicole Gaudelli, the Associate Director and Head of Gene Editing Technology at Beam Therapeutics...insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a problem. Therefore...editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA to contain ...editors are precise molecular tools, they do have undesired off-target editing effects. ABEs have been described...off-target editing frequencies. For applications that require high DNA editing specificity, for example for potential...

  20. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...extra level of safety, they also require a fourth plasmid. The requirement for additional DNA can impact ...third generation systems such that it no longer requires the viral transactivator, Tat. For more information...increasing the number of recombination events that are required to generate RCL. To overcome the titering hurdles...unconcentrated prep. With this in mind, if the experiment requires a certain volume of virus, consider scaling up...larger vessel, such as a 3 or 5-layer flask will require some initial optimization; cell number, media volume... method is relatively inexpensive and does not require an ultracentrifuge, PEG will precipitate all proteins... may not be the best method when pure virus is required downstream. When assessing lentiviral titer, don't...

  21. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    ...expand applications that require very specific targeting, like homology directed repair and base editing...order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence...David Liu’s lab, have gone one step further, using directed evolution to create xCas9, an enzyme recognizing...more complicated if you’d like to use homology directed repair to make precise edits or knock-ins. Editing...other species, like S. aureus and N. meningitidis require different PAM sites than SpCas9, in this case, ...broader PAM flexibility. Creating xCas9 through directed evolution Hu et al. used phage-assisted evolution...about CRISPR Base Editing Learn about Homology Directed Repair Resources on Addgene.org Check out our...

  22. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...users to generate a plasmid containing a direct fusion of the desired gRNA to the U6 promoter, and a second...genomic site and, using the endogenous homology directed repair pathway, a new user-defined DNA sequence...designs either oligos or synthetic DNA for the desired gRNA target sequence, as well as the 5’ and 3’ ... and a Cas9 expression plasmid will insert the desired genetic tag and marker sequence into the targeted... the insertion, and 3) a vector containing the desired tag, which can be obtained as a universal donor...expression vector. They can also be introduced directly into a Drosophila S2 cell line stably expressing...homology to the gene of interest to promote homology directed repair after a CRISPR/Cas9 induced break. Learn...

  23. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    ...kind of resources - including your time - will be required to produce an outcome you’re happy with. Know ...semester-long after school program? Comparing your desired outcome to the limitations and potential of your... communications below!   Format Resource Requirements Pros Cons Video Medium to high Uses visuals.... Easy access from smartphones with low data requirements.  No visuals. Variety of hosting platforms can...distribution.  Static. Can be difficult to convey an entire story or context via still images. Some things ... Digital or print distribution.  Static. Often requires both artistic and scientific training. Cartoons...media type to another is cognitively demanding. Requires multiple areas of expertise.  Social Media ...

  24. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    ...quantity of DNA that is required and, as a consequence, also dictate the required sizes of the bacterial...extraction. Restriction enzyme based approaches can require several hundreds of nanograms or micrograms of ...Gibson assembly or Golden gate assembly use a more direct approach, allowing scientists to clone from a smaller...extraction. Figure 2: Use cloning grade DNA for direct cloning of your gene of interest into a plasmid... plasmid in mammalian cells and it will likely require micrograms of plasmid DNA for each reaction. As...chemical contaminants, these DNA samples must also be entirely endotoxin free. Endotoxin is a lipopolysaccharide...

  25. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ... beginning of the coding sequence to delete the entire gene? Perhaps you are more interested in the c-...expression cassette. The target sgRNA can be cloned directionally into the BbsI site. (c) The pCAG-EGxxFP target...the two halves of EGFP to recombine by homology directed repair, and resulting in the expression of EGFP...component of the system. However, it is easier to directly inject the pX330 plasmids used in the validation...Working with plasmid DNA is much easier and doesn’t require the extra precautions needed when dealing with ...they are born, it’s time to screen them for your desired edit. Good news! Remember the primers you designed...genotyping), but bear in mind that this method will require you to house mice that may have no mutation, costing...

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

    Type
    Blog Post
    ...don’t require the organism to be specified and are truly organism agnostic. Other tools require an organism...Multi-locus asseEENdb database ssment would be required to fully evaluate off-targeting, but currently...design. These databases are still young and will require a collaborative effort from the community in order... Advanced functions: These functions are not entirely necessary but should be considered extremely useful...available mostly work on any computer but generally require working knowledge of the command line or scripting...scripting. Input flexibility: Software tools require some kind of data input in order to generate results...it takes authors to add a genome to the tool’s repertoire. Organism agnostic tools are the better solution...

  27. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ...doesn’t require a functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-... notably CD4+ T-cells, and eventually leads to acquired immunodeficiency syndrome (AIDS) in untreated ...infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma... to survive, but preventing re-infection would require sustained expression of Cas9/gRNAs, which could...at the level of protein structure, CRISPR gRNAs require DNA sequence-specific binding. Patients’ HIV-1 ... and kill” strategy may be a better option than direct cleavage. Despite the potential difficulties in...Putatunda R, Young WB, Khalili K, Hu W. CRISPR/gRNA-directed synergistic activation mediator (SAM) induces ...

  28. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...bypasses the 5′ mRNA cap required for eukaryotic systems and instead directly engages the ribosome for... Good tools for measuring intracellular pH are required in order to determine the relationships between...fluorophore is pH-dependent. Signal is simultaneously acquired from the the fluorescent and luminescent siganls... a gene-specific forward primer containing the desired restriction site (the MCS has several options) ...specified order using Multisite Gateway cloning. A desired insert is cloned using traditional restriction ...vectors in a Multisite Gateway reaction with a desired destination vector expressing a separate reporter...MluI, XhoI or SalI sites must be removed by site-directed mutagenesis in order to use the module as a building...

  29. CASTing Off for New Shores in Human Genome Editing

    Type
    Blog Post
    ...be overcome by directed evolution using bacteriophages. Researchers moved a gene required for phage propagation...breaks and can deliver large sequences and even entire genes. Figure 1: Mechanism of CAST systems...eukaryotic cells — at least, until they met the directed evolution expertise of the Samuel Sternberg and...highly tailored treatments to specific mutations require individualized optimizations, safety testing, and...experiments, PASSIGE had higher efficiency, but evoCAST required less optimization for new targets and produced.... (2019). Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature, 571(7764), ...

  30. Antibodies 101: Affinity Tags

    Type
    Blog Post
    ...primarily used for direct visualization while epitope tags are used for both direct and indirect visualization...must be performed after primary purification and requires a second clean up step to remove the cleaved tag...Also, purifying GST by affinity chromatography requires proper folding of the tag, which prevents insoluble.... Mammalian purification is still possible but requires optimization and often more stringent washing....especially if downstream antibody detection is desired… and yes, we have an antibody for that!   ...

  31. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    ...independent, it does not require purification of insert or vector products, nor does it require the use of specific...purification, and, most importantly, excessive time requirements. FastCloning is a simpler yet reliable cloning...Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning Choose Restriction...templates. Digestion products are then transformed directly into chemically competent E. coli and transformants...competent E. coli for this procedure to work properly. Directly transform chemically competent cells with the ...Addgene Blog Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background...

  32. CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

    Type
    Blog Post
    ...materials. Problems with Chip-seq ChIP-seq typically requires a large amount of input material, cells or tissue...experiment can be a major technical hurdle.   ChIP-seq requires  cross-linking during an initial fixation step...sites when in fact they are just non-specific or indirectly interacting. The CUT&RUN method Enter CUT&RUN... the University of Washington. CUT&RUN doesn’t require fixation and significantly decreases background...wing as a model. A typical ChIP-Seq experiment requires around 1,000 wings. Collecting that much tissue...controlled by the addition of calcium, which MNase requires for its nuclease activity and is chelated from...cells or when a high degree of signal to noise is desired. It also provides an excellent additional measure...

  33. Management for Scientists: Managing vs. Leading

    Type
    Blog Post
    ...ensuring good communication. A manager must both hire and fire to make sure the team is the best it can be....organization and is also one of the most common criteria required for a promotion. Yet little explanation is given...quality. Their work involves coordination and can require technical mastery. Scientists need to be doers ...successful because you are not fulfilling the requirements of the current task. Most of us start as doers...may be happy with a primary role of doer for your entire career. Scientists train as doers and often maintain...download the Addgene Management for Scientists Ebook.  Fire, Snowball, Mask: How Leaders Spark and Sustain Change...

  34. Even more elegant: Single injection CRISPR/Cas9 in C. elegans

    Type
    Blog Post
    ...just one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth...signaling a new era in C. elegans research. Homology directed repair (HDR), which enables insertion of custom... elegans/CRISPR experiments, but this strategy required the use of unhealthy, difficult-to-inject unc-...injected 60-80 animals per gene, they obtained the desired insertions on the first attempt for 6/7 genes. ...Dickinson et al. have developed a modular system that requires very little hands-on labor and could facilitate...Better Cas9 Editing Efficiency CRISPR 101: Homology Directed Repair  ...

  35. Advice for Scientists Starting a Lab

    Type
    Blog Post
    ...select first hires who are well-suited, productive, and fit well with your goals. These first hires will set...set the tone and atmosphere that future hires will look up to." She also recommends to seek out advice...hiring process. Read Genevive Bjorn's "Ready, Set, Hire" on Nature Jobs for some quick advice and resources...surprised by the variety of skill sets she needed to acquire as a professor. “Being a PI is more than just experiments...recognize that every graduate student or postdoc may require different types of mentoring. Some mentees prefer...lab and selectively picking the first couple of hires to build a strong, collaborative team. And take ...

  36. Why and how to start studying chromatin (with Addgene's help)

    Type
    Blog Post
    ...Chromatin regulation is complex and influences--directly or indirectly--many other areas of biological research...looking for on this list, please consider searching directly on our website. In-exhaustive Chromatin-related... might be useful for your work. The links below direct you to pages containing lists of plasmids from ...publications. You can request all of these plasmids directly from Addgene. Note that there are plasmids from...recombination. Gong F, Chiu LY, Cox B, Aymard F, Clouaire T, Leung JW, Cammarata M, Perez M, Agarwal P, ...techniques, the posts below may steer you in the right direction! CAPTURE-ing Chromatin Interactions: Using CRISPR-dCas9...

  37. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ...nucleotides within a defined region of a genome. Require stringent bacterial growth conditions. Expression...capable of mutagenesis in E. Coli, but this approach requires special bacterial growth conditions in order to...Phage Assisted Continuous Evolution (PACE) method requires constant turbidity of the culture, so bacteria...introduce mutations. However, by using nCas9 to direct PolI3m to a particular genomic loci, EvolvR allows...mutagenesis of a site of interest. A gRNA is used to direct the PolI3M-nCas9 complex to a DNA site of interest...PolI) that is fused to a nicking Cas9. A guide directs Cas9 to a site of interest, where it nicks its ...lambda red recombineering of bacteria Use site directed mutagenesis to generate specific mutations in ...

  38. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    ...advancements and the noteworthy work of fiction that it inspired. CAUTION: Spoilers alert! Pre-DNA Era  H.G. ...In the 1960s, Har Gobind Khorana, Marshall W. Nirenberg, and Robert W. Holley cracked the genetic code...and the rise of killer machines that exterminate entire humanity were the two most popular topics that ... agricultural products. But it also made SciFi director Ridley Scott wonder what would happen if these...engineering to exploit human beings. Ridley Scott directed Blade Runner (1982). It is a dystopian film that... double strand break. These breaks can then be repaired by homologous recombination with a repair template...mutations in the genome. They fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate...

  39. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...This post was contributed by Erik Snapp, the Director of Student and Postdoctoral Programs at theJanelia...writing can take weeks to a couple of months and requires that all of the experiments have been completed... that fund your research, guides the research directions of the lab, gives you constructive feedback on...Furthermore, Group Leader position applications require 3 letters of reference- the graduate school mentor...thoughtful engaging talk takes significant effort. It requires evaluation of all of the data and any recent relevant...new pieces of data, your talk will probably not inspire you or the audience. Ask yourself: What is the...becoming a group leader is that people are typically hired for a job that is orthogonal to their scientific...

  40. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent...illumination, TIRF or spinning disk confocal use entirely different excitation power regimes. Therefore,...that a photostable fluorescent protein is a key requirement for quantitative live-cell imaging, it is important...photostability of the fluorescent proteins can be directly compared. An example of a photostability measurement...proteins or localized fusion proteins and do not require dedicated plasmids or constructs. It should be ...imaging would be mTurquoise2, based on the data acquired at low excitation powers. Note, however, that,...Plasmids for Subcellular Localization Browse the entire Fluorescent Protein Collection ...
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