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We narrowed to 606 results for: abo.2

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  1. New England Biolabs Cell-Imaging Plasmid Collection

    Type
    Collection
    ...transient mammalian expression 101126 pACP-tag (m)- 2 Vector ACP/MCP-tag Empty backbone for stable and transient...available Non-toxic to living cells To learn more about this system, visit the New England Biolabs website...

  2. Luciferase Plasmid Collection

    Type
    Collection
    ...fusing NanoLuc® and Venus fluorophore to Troponin C Ca 2+ binding domain. Nano-lanterns , Enhanced Nano-lanterns...transfection Feng Zhang 21471 pLenti PGK V5-LUC Neo (w623-2) Firefly PGK Lentiviral expression of firefly luciferase...Plasmids are Gateway cloning compatible. Learn more about DULIP in our 27 Hot Plasmids from 2016 blog post...circularly permuted Venus fluorophore. Learn more about pHlash sensor in our 28 Hot Plasmid Technologies... with the original STARR-seq method. Learn more about STARR-seq in our 27 Hot Plasmids from 2016 blog ...

  3. Who Gives a Tweet? 9 Facts About Scientists on Twitter

    Type
    Blog Post
    ...Occupational Classification System  and Wikipedia. 2. Social and computer scientists are overrepresented...international Twitter users were included in the dataset; 2) users not part of a scientific Twitter list were ...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of scientist...use Twitter professionally to network, learn more about alternative careers for scientists, and share cool....” This paper addresses the following questions about scientists on Twitter: who are they? What do they...

  4. Brain Initiative Collection

    Type
    Collection
    ...Gordon Fishell 83899-AAV1 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAV9 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAVrg pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...particles that are suitable for in vivo use. Learn more about our viral service ID Viral Prep Description PI 65417...well as by our trusted partner labs. Learn more about our antibodies ID Recombinant Antibody Reactivity...

  5. A Look Back at One Year of Plasmid Sharing for COVID-19 Research

    Type
    Blog Post
    ...SARS-CoV-2 spike pseudotyping, visit the SARS-CoV-2 Pseudotyped Virus collection page. SARS-CoV-2 detection...of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV-2 viral proteins...neutralizing the different SARS-CoV-2 spike variants. You can learn more about their research in this article...resources on the Addgene blog Learn more about mapping the SARS-CoV-2 - human protein-protein interaction ...labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from Maya Kostman... protein, variants, and pseudotyping The SARS-CoV-2 virus depends on its spike protein to bind ACE2, the...of infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab. Jesse Bloom’s lab published...

  6. Crowdfight, a Platform to Boost Scientific Collaboration During COVID-19 and Beyond

    Type
    Blog Post
    ...requests every day.    Figure 2: When a researcher needs a collaborator for a specific task, Crowdfight...mammal called alpaca could neutralize the SARS-CoV-2 virus. But while the early results were promising,...Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert’s suitability and availability...our blog post about remote collaborations Read our Transferable Skills Guide article about external collaborations...needed. In the example above, for example, we fostered a formal scientific collaboration between two research...project. We think that these collaborations add enormous value. A discussion about a protocol can save weeks..., the researchers did not have access to a P3 laboratory (BSL-3), a high-security facility needed to perform...

  7. Reagent Repositories Are Speeding up Science During the Pandemic

    Type
    Blog Post
    ...regards to SARS-CoV-2, this mouse strain is regaining popularity. The Jackson Laboratory, a nonprofit research...SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies...fast access to the tools required to study SARS-CoV-2, but with many academic research labs closed during...biobanks, are crucial for the advancement of SARS-CoV-2 research because they provide rapid access to validated...Many of the reagents used to investigate SARS-CoV-2 were developed for broad use and are now important...susceptibility of cells from different organisms to SARS-CoV-2 (Chu et al., 2020). An additional cell line used in...that science is truly a global endeavor. Case study 2: Plasmids Another example of reagents that have broad...

  8. New and Upcoming Viral Vectors - May 2020

    Type
    Blog Post
    ...pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a... SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins at...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro...months: Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP ...Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2  Additional resources on the Addgene blog Read...vector name and serotype you’d like to be notified about. Chemogenetics Plasmid Serotype Name 44362 ...

  9. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...because: 1) their genomes are easy to manipulate, 2) they have long-term expression; and 3) they have ... On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains ...stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction...cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e. tet-off transactivator...

  10. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    ...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...relying on raster scanning as is done in confocal or 2-photon microscopes. Total Internal Reflection Microscopy...

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

    Type
    Blog Post
    ...coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12. Cold Spring Harbor Laboratory. https://doi.org...Based Detection of SARS-CoV-2 Genetic Material. Cold Spring Harbor Laboratory. https://doi.org/10.1101/2020.04.20.052159... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...

  12. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    ...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...blog posts Learn about other COVID-19 plasmids available at Addgene Read about SARS-CoV-2 detection methods...Bioworks to synthesize and share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility...optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression...

  13. "What Makes a Good Mentor?" and 6 More FAQs About Science Mentoring

    Type
    Blog Post
    ...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and...be challenging – is advice and guidance from only 2 people enough?  For many years I’ve been organizing...Connie Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this... answers to the top questions scientists ask me about mentoring. Here they are: What makes a good Mentor... Where can I find a Mentor? What is this I hear about “group” or “peer” mentoring? How do I ask someone...a good science mentor? I started a conversation about many of these topics with the researchers who joined...often one that devotes time and energy to thinking about how to be a great Mentor.    Check Out Angela Depace's...

  14. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    ...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...details. Our set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

  15. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    ...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ... SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple...viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr... binding ACE2 is the target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the...and a barcode, and is designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface...

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

    Type
    Blog Post
    ... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D.... The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely...ultracentrifugation [2]. Some affinity assays used in the papers describing the FPs above including molecular... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest fused to a dimerizing... designed to detect resulting in false positives [2, 4]. Solutions to the polymer problem There are some...

  17. Antibody Neutralization Response Against Pseudoviruses Expressing SARS-CoV-2 Spike Protein Variants

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    Blog Post
    ...months, we’ve seen the emergence of several SARS-CoV-2 variants coincide with increased vaccination efforts...They were able to tweak this assay to study SARS-CoV-2 spike variants. “We created a series of spike expressing...relative ability to neutralize.”   Figure 2: Sera used in the study came from individuals who ...This sera was used in the high-throughput SARS-CoV-2 pseudovirus neutralization assay. Neutralization was...viruses are quite genetically distinct from SARS-CoV-2 and its variants. Yet, the B.1.351 variant, which ...effectiveness of current vaccines against SARS-CoV-2 variants, this study was the first to compare so many... de Oliveira T, Sigal A (2021) Escape of SARS-CoV-2 501Y.V2 from neutralization by convalescent plasma...

  19. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    ...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...Additional Resources on the Addgene Blog Learn More about Photoactivatable Fluorescent Proteins Control Protein...

  20. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    ...condition 1” than “condition 2.” By eye, the loading control appears about the same between the two conditions...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

  21. Antibodies 101: Fab Fragments

    Type
    Blog Post
    ...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

  22. Addgene's Top Blog Posts from 2020

    Type
    Blog Post
    ...CRISPR to detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...highlighted reagent repositories and other collaborative efforts in the scientific community to speed COVID-19 research...store my plasmid in TE, Tris, or water? And what about temperature? Find the details in this blog post....dominates DNA quantification discussions, but what about the other methods to quantify DNA? Here we cover...

  23. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...completely inert, but others can cause diseases. Nervous about handling viruses? That’s okay! In this article we...

  24. Viral Vectors 101: Pseudotyping

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    Blog Post
    ...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ...SARS-CoV-2 virus. Scientists have been using similar pseudotyping methods with other viruses in a BSL-2 lab... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid...example, production of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl...for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 12...Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116 Gutierrez-Guerrero...

  25. Uncertainty about Labor Law Brings More Uncertainty to Postdoc Wages

    Type
    Blog Post
    ...that increase cancelled. Rounding out the remainder, 2% of postdocs who weren’t going to get a salary increase...with their plans, and 17 have cancelled their plans. 2 of those raising - the University of Illinois and ...Previous Article: Changing Labor Laws Bring Increased Postdoc Wages Read about the Advocating for Science... of $47,476 per year, under updates to the Fair Labor Standards Act (FLSA). This was due to the threshold...postdocs - and in some cases, faculty - have found out about what their institution is doing from Future of Research...Science Symposium and Workshop Learn about a Variety of Science Careers Resources on Addgene.org Find Career...

  26. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    ...same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position. ...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of...“I hope it isn’t degraded” when scientists talk about their precious RNA samples. RNA is inherently less...efficacy as well as the unnecessary stress of worrying about the status of your RNA. gRNAs modifications are ...Some of the RNA stabilizing modifications listed above were developed before CRISPR-Cas9, for use in applications...

  27. Great Results Start with Great Standard Curves

    Type
    Blog Post
    ... Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series that ranges...series ranging from 1:2 to 1:32. Each individual step of the series is consistent at 1:2. When preparing a.... An example of this is shown in Figure 2.   Figure 2: The concentration of a serially diluted... ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration of ...accuracy of the curve, avoid pipetting small volumes (< 2 µL) or volumes too large for a standard micropipette...by replicate values provides useful information about the accuracy of the curve, which we will discuss...many scientific applications users aim for an R2 above 0.95.    Additional considerations When running ...

  28. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...

  29. Typing CRISPR Systems

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    Blog Post
    ...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

  30. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...inserted GFP is expressed (knocked in). As seen in the above example, you can remove a floxed resistance gene...

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

    Type
    Blog Post
    ...designed (listed above) worked well and I got clear bands using the conditions above. 2. Design your primers...expression, but I didn’t. 2. In the original publication they see GFP expression in about 20% of their Cas9-expressing...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn... appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this...

  32. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ...between a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-resolution...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express... Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., ...

  33. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for...information about the contents of the autophagosome nor does it provide information about what is being...higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella... (as opposed to giving more fine-grained detail about what proteins or organelles are being degraded) ...

  34. Hot Plasmids - October 2022

    Type
    Blog Post
    ...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...Brian O'Neill  The François St-Pierre lab and collaborators have successfully developed an improved voltage...

  35. The Materials Science of Optogenetics Experiments

    Type
    Blog Post
    ...Laser stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor...These lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,... the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...distance so ideal implant placement should be about 0.4-0.5mm above the site of your stimulation. The scatter...

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

    Type
    Blog Post
    ... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ... cloning Read our Plasmids 101 blog posts Learn about the differences between transformation, transduction...

  37. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,... virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant and/or producer...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...dehydration (Howard & Harvey, 2017). You can learn more about common AAV applications by reading our Viral Vectors...

  38. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...image above). A second universal primer is used to synthesize a complementary mutant strand (step 2 and ... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

  39. Tetracycline Inducible Expression

    Type
    Collection
    ...expression tTA Off Verma 26803 pEnt L1L3 EF1a-tTA-2 Contains Gateway L1L3 sites and EF1α promoter driving... or shRNA; see article for detailed information about cloning and additional plasmids tetR-KRAB On Aebischer...

  40. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...republished here with permission. Quilt is a collaborative database for genomics. In this article, Quilt...tri_methylation In the next section we’ll apply the above formula to a real-world experiment. We’ll start ...
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