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We narrowed to 791 results for: Cre

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  1. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    Published
    Aug. 18, 2016, 2:30 p.m.
    ...in therapeutics, as it can increase gene expression at certain loci to create changes in phenotypes. For... storm. While Cas9 has been most widely used to create specific edits in DNA, there has also been significant...inducible upon environmental triggers. This is done by creating a split dCas9 protein that can reassemble when...the best uses for Cas9 activators is in genetic screening. gRNAs targeting every gene in the human genome...interactions. Additionally, large scale CRISPR-based screens can be used to gain insight into protein structure...focused advancing scientific discovery through the creation of innovative technologies. To date his group ...of cancer therapeutic targets using CRISPR–Cas9 screens. Nature 568:511–516 . https://doi.org/10.1038/s41586...

  2. CRISPR 101: Cas9 Nickase Design and Homology Directed Repair

    Type
    Blog Post
    Published
    March 15, 2018, 12:59 p.m.
    ...nuclease domains, researchers created the CRISPR nickase. Nickases create a single-strand rather than a...generated by the nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design...Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving nickase. Instead of...strands bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs...other? The gRNAs must target different strands to create a DSB, but this can be accomplished with either...an individual gRNA with WT Cas9, repair levels decrease rapidly 10 bp from the cut site. So if you can... you can’t obtain high HDR efficiency. Nickases create a staggered cut - could this system mediate repair...

  3. Fluorescent Biosensors for Measuring Autophagic Flux

    Type
    Blog Post
    Published
    Jan. 22, 2019, 2:41 p.m.
    ...autophagosomes with lysosomes leads to an increase in yellow puncta but a decrease in red puncta. Find the mRFP-...RFP ratio: a decrease in GFP fluorescence relative to RFP fluorescence suggests an increase in autophagic...flux, while an increase in GFP fluorescence relative to RFP fluorescence suggests a decrease in autophagic...yeast or the lysosome in mammalian cells. This decrease in pH quenches SEP but not DsRed, leading to emission...Therefore, the formation of autophagosomes leads to an increase of yellow puncta (GFF positive/RFP positive), ...fusion of an autophagosome with a lysosome. An increase in autophagy leads to more yellow and red puncta...

  4. Antibodies 101: Single Chain Fragment Variables (scFvs)

    Type
    Blog Post
    Published
    June 3, 2021, 1:15 p.m.
    ... rounds of screening. Created with Biorender.com.   Some phage display libraries screen antibody genes...serine with dispersed hydrophilic residues for increased solubility (Monnier et al., 2013). The linker ...expression systems. scFvs are also small enough to be screened for with in vitro display methods such as phage...applications where longer retention times often increase therapeutic efficacy (Ahmed et al., 2012). How...and VH sequences. Phage display also allows for screening scFv generated from the B cell antibody genes ...the ways antibodies are. scFvs are even used to screen for new antibodies. To do this, antibody variable...variable domain sequences are expressed as scFvs and screened with in vitro assays to select for strong binders...

  5. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ... are isolated from the resulting colonies, and screened for the desired modification. Finally, the positive...final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of interest...multimerization (purple) which is evident by an increase in the size of the lower band (portion of the ...mutation). Similar results can be obtained with PCR screening using the primers specified by the arrowheads ... vastly facilitates the subsequent process of screening for succesfully mutated clones. Forward and reverse...Template A high purity plasmid prep significantly increases the success rate of site directed mutagenesis....secondary structures of the DNA template, and may also decrease the annealing temp of the primers. Because you...

  6. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    Published
    May 26, 2020, 1:15 p.m.
    ...14 and 21 to create GEmCherry2. The Denham lab made several other small changes creating 2 other versions...Cas9 activity assays. Recently scientists have created CRISPR fluorescent reporter assays to quantify ...CRISPR/Cas-stimulated HDR activity) The Madison lab created SRIRACCHA in 2017 to enrich and identify desired...H2B-GFP. This visual read out allows for easy screening of different gRNAs by simply changing the gRNA...plasmid that contains Cas9. The Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where... H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous indel edits when compared... sites and activity (Knudsen et al., 2018).  To create GEmCherry2, the Denham lab first incorporated an...

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

    Type
    Blog Post
    Published
    Jan. 11, 2017, 3:30 p.m.
    ... the injunction. While much of this decrease is due to an increase in the number of universities’ whose...postdocs who were expecting a salary increase have had that increase cancelled. Rounding out the remainder...payment for working more than 40 hours per week increasing from $23,660 to $47,476 per year. This post discusses... of postdocs who weren’t going to get a salary increase prior to the injunction still won’t after the ...issues such as childcare using money from their increased salaries. In addition, salary uncertainty may ... costs of living would get much better salary increases that those in less densely populated areas. This...Gary's Previous Article: Changing Labor Laws Bring Increased Postdoc Wages Read about the Advocating for Science...

  8. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    Published
    Aug. 27, 2015, 2:30 p.m.
    ... of interest. Although efficiency may decrease with an increased number of fragments, or the ligation ... cleave outside of their recognition sequence, creating four base flanking overhangs. Since these overhangs... fragments, these problems can be overcome by screening a higher number of potential clones. Golden Gate...the ordered assembly of multiple DNA fragments to create TAL effector nucleases. These plasmids were designed...This cloning strategy not only makes it easy to create a single gRNA-expressing plasmid, but it can also...fragment: PCR-based amplification can be used to create silent point mutations at internal recognition ...Whether you are using the Golden Gate method to create CRISPR/Cas9 constructs, assemble standard plasmids...

  9. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    Published
    May 24, 2018, 1:20 p.m.
    ...challenging for amateur scientists. Planarians, wormy creatures commonly found in freshwater ponds, provide a ...promoters to allow for bidirectional transcription to create dsRNA. Students can produce DNA templates via PCR... PCR off of the same plasmid and use these to create riboprobes for in situ hybridization experiments.... as how to perform worm amputations. They also created an online companion resource called “cutting class...within an embryo or tissue. Once the riboprobes are created, the WISH protocol involves preparation of the ...as a result, these cells turn blue. Students can create synthetic riboprobes off of linearized DNA templates...from their organism of interest, use the RNA to create a complementary DNA (cDNA), and then use PCR and...

  10. 3D Printing Meets CRISPR Cas9

    Type
    Blog Post
    Published
    Oct. 17, 2017, 12:55 p.m.
    ...protein in your hand? At 3D Molecular Designs, we create physical models of proteins using 3D printing technology... real”.  We sometimes forget that the models we create are around 30 million times larger than the real...were soon developed that allowed researchers to create stunning, interactive images of their proteins....physical models of protein structure? We have been creating physical models of proteins for researchers for...educators and students use molecular models?   We created the Center for BioMolecular Modeling (CBM) in 1999... 1999 with the expressed purpose of creating physical models of proteins and other molecular structures...researchers to learn about their research while creating physical models of proteins that are being investigated...

  11. Microbes: Look, Listen, and Tell

    Type
    Blog Post
    Published
    Aug. 28, 2018, 12:04 p.m.
    ...in vivo: acoustic reporter genes’ on our blog. Creative approaches to presenting the microbial world ...bacteria in the lab, I was excited to learn about creative approaches to visualize bacteria in an educational...educational setting. The Symposium on ‘Creative Approaches to Presenting the Microbial World’ showcased complex... comics (Dr. Sanja Saftic) to educational gifs created by the Amoeba Sisters. Here’s one example of how...lactose-around-we-can-make-some-enzymes-for   The take home message was that being creative and telling a story makes scientific topics both... fascinating to see the living art designed by creative minds with a (I assume) very steady hand. By connecting...connecting art and science, a fascinating interface is created that produces loads of inspiration for the current...

  12. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    Published
    Nov. 29, 2022, 2:15 p.m.
    ...genome-wide screens, introducing bias into all work based on these and any other CRISPR screens using the..., race. Many researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic...targets and related biomarkers . Data from such screens are often uploaded to the Cancer Dependency Map...Boehm labs pulled data from more than 1000 CRISPR screens in DepMap to see if they could identify the ancestry... American, Africa, or South Asian) used in the screens based on the cells’ germline genetic variations...about 90% of the cell lines used in DepMap CRISPR screens are of either European or East Asian ancestry, ...actually reference?  The CRISPR guides used in the screens they analyzed were designed to have a perfect match...

  13. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    ...breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting...type to subtype to variant (shown in italics). Created with BioRender.com.   Class 1 versus class ...underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class 1 CRISPR systems are less popular...Cascade. As you might imagine, this is handy for creating large genomic deletions. Type I systems are also...with Cas3 (pink). Complex shown is from type I-E. Created with BioRender.com.   Type III Type III is ...most engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision...CRISPR-Cas9 mechanism, representing type II-A. Created with BioRender.com.   Type V Type V uses Cas12...

  14. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    Published
    May 2, 2019, 12:41 p.m.
    ... progress on the NSF “G to P grand challenge.” Creating a productive, new scientific community is no small...in particular, that they all share the plasmids created under this programs via Addgene. New organisms ... organisms) -  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project...crisis. I like how they are using protocols.io to create a protocol community! Asclepias syriaca and Asclepias...used to study aspects of cognition. Spiralians - Crepidula atrasolea (Jonathan Henry) This black footed slipper...can express fluorescent proteins. This lab has created an automated tank for feeding and maintenance that...real bottleneck. New gif for #MolluscMonday: #Crepidula atrasolea juvenile, light microscope with polarizing...

  15. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    Published
    April 11, 2017, 2:30 p.m.
    ...and Carrot, and further means to increase aptamer stability and decrease background in vivo. The nature ...enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide... visualization techniques. In addition, their incredible versatility opens aptamers for use in many entirely...aptabeacons (or aptatags) were born. Aptabeacons can be created in many different ways, but the general premise...that bound the compound malachite green (MG) to create a non-protein based visualization technique. Ultimately... knowledge of the nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing... RNA aptamer that would bind the fluorogen and increase its fluorescence, essentially mimicking GFP.  ...

  16. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    Published
    Jan. 12, 2017, 3:30 p.m.
    ...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain...Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination of an attB-flanked...attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the insert into an...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the inset into...vector will recombine with the attL-entry clone to create the expression clone.    Figure 6: Generating...between the entry clone and destination vector creates two products: the desires expression clone and ...

  17. Bioinformatics at Addgene

    Type
    Blog Post
    Published
    July 14, 2022, 1:15 p.m.
    ... we grew. As the number of deposited plasmids increased, the accompanying sequencing data collection and...bioinformatics workflow management system As novices to creating a robust data pipeline that would be reliable ...community of Nextflow supporters existed who had created a shared resource of pipelines for genomic data... with others without any complicated setup. We created, tested, and debugged each of our pipeline steps...running the pipeline locally and in the cloud. Creating a bioinformatics workflow in Nextflow Establishing...scripting language that can be executed on Linux, we created our first process by simply copying and pasting...was working as expected, we moved forward with creating the next process and linking the output from the...

  18. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    Published
    Jan. 24, 2023, 2:15 p.m.
    ...vesicles with nonlinear contrast. Increasing expression and decreasing genetic footprint We took a cue ...heterologously by bacteria inside the body. So we screened gas vesicles gene clusters from a diverse set ...expression heterogeneity required single cell screening to isolate a clonal cell line capable of producing... expression to appreciable levels. We wanted to create second-generation mARGs with higher expression,...reduced the number of copies of gvpA needed to increase expression. We were able to express Anabaena ...expression leads to greater amount of gas vesicles and increased ultrasound contrast. In all the cell lines we ...their tracking with ultrasound imaging (Fig. 2d). Creating a cell line can be a time-intensive endeavor. ...

  19. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    Published
    Feb. 22, 2017, 3:30 p.m.
    ...polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased concentration...PCR reaction is created by making some or all of the following modifications: 1) increased concentration...saturation mutagenesis Several methods can be used to create mutational scanning libraries. While each of these...Therefore, Gibson assembly is better suited for creating small, focused scanning libraries. PFunkel:...library that’s ready to be tested in a functional screen of your liking. This method is similar to site-directed... screen...

  20. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    Published
    June 27, 2024, 1:15 p.m.
    ...as a stepwise decrease in signal strength in a flow plot. d = days post treatment. Created using biorender.com...binding and (b) antibody host species interactions. Created using biorender.com. Another way of indirect...1b). As above, the advantage of this system is increased flexibility for panel design. Another perk is ...can be bound by several secondary antibodies, increasing the fluorescence signal intensity. However, you...cell, CD4+ T cell, dendritic cell, CD8+ T cell. Created using biorender.com.   Intracellular labeling...cytosolic proteins, as well as proteins of the secretory pathway. Those can include cytokines and chemokines...bind to their targets in the cytosol and nucleus. Created using biorender.com.   Dye stains Dye stains...

  21. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    Published
    Dec. 6, 2022, 2:15 p.m.
    ...was identified from a high-throughput screen, selected for increased voltage response, brightness, and photostability... They use a unique and powerful high-throughput screen to select variants based on performance metrics...resulting JEDI-2P has faster response kinetics and increased brightness, sensitivity, and photostability under... be generalized to other RTKs of interest, they create sensors for FGFR1, EGFR and ErbB2. And orthogonal...developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite...cellular concentrations of lactate. The microfluidic screening platform developed here is also a promising approach...Figure 6: A) Schematic of BeadScan platform for screening candidate biosensor variants for performance under...

  22. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    Published
    Sept. 14, 2016, 2:30 p.m.
    ...greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of plasmids...markers built into all plasmids in the kit help decrease false positives observed in the cloning process...protein combinatorial library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid ...set 3000 Series Confronted with the problem of creating vectors for predictable protein expression in ...bacteria, researchers at the BIOFAB and the Endy Lab created this kit. The plasmids within it contain a variety...assembly. PhD student Dejan Gagoski used SITS to create the pCell-Free vectors, Gateway-compatible backbones...these different modular repeat segments, you can create a PUF protein that binds your RNA sequence of interest...

  23. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    Published
    Nov. 25, 2015, 4 p.m.
    ...topics discussed during the symposium will prove incredibly important to the stability of the biological ...align trainee expectations with job outcomes, and increase diversity in the academic research environment...these problems and some that stuck out to me were: Decrease the Number of Postdocs De-incentivize going into... more services based on what they're spending. Increase the number of science masters programs to prepare...postdoc salaries as opposed to Staff Scientists Increase the Starting Salary for Postdocs to $50,000 (the...salary as reported by Paula Stephan is $40,000) Increase Knowledge about Postdoc and Graduate Student Career...was highlighted during the conference that just increasing funding for academic research will not solve ...

  24. Viral Vectors 101: What is a Virus (and a Viral Vector)?

    Type
    Blog Post
    Published
    Aug. 18, 2023, 4:15 p.m.
    ...payload to a cell but cannot create new particles. As a bonus, this increases the carrying capacity of the...non-enveloped virus (left) and an enveloped virus (right). Created using biorender.com   If you’re new to talking ... Figure 2: Baltimore replication classes. Image credit: Louten, 2016 Viruses with DNA genomes can either...After a virion infects a cell, the single particle creates many copies of itself, sometimes up to millions...the risk of infection — researchers were able to create far safer viral vectors that can be used in BSL... the viral vector, increasing both safety and usefulness of the tool. In fact, most gutted genomes have...have as much genome removed as possible, just to increase the viral vector’s carrying capacity. In order...

  25. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ..., the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance... initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3-fold... Figure 1: An overview of prime editing. Created with BioRender.com. How does it work? First... between the template and primer binding site, increase processivity, or improve thermostability. What...fold. However, the process of double nicking increases indel formation slightly. Designing the sgRNA with...inventors kept optimizing it. Using a pooled CRISPRi screen, the Liu lab discovered that cellular mismatch ...addresses the problem of RNA stability. Using a CRISPRi screen, they identified a protein that enhances prime ...

  26. Fluorescent Protein Travel Awards - Protein Variants, a Serotonin Sensor, and an Artificial Leaf Replica System

    Type
    Blog Post
    Published
    June 4, 2019, 1:02 p.m.
    ...site-saturation mutagenesis, and medium-throughput screening, she increased the affinity of the sensor for serotonin...overwhelming response and were excited to see the many creative uses of fluorescent proteins. We’re happy to announce...thousands of protein variants simultaneously  With increasing sequencing power, scientists are able to quickly... protein-coding pathogenic human variants are decreased in protein abundance, possibly due to misfolding...neuromodulator, and evidence suggests that it is incredibly important for nearly every aspect of life.” Unger...system. “This bottom-up approach will allow me to create a fully controllable and reproducible set up,” ...explains. Developing an artificial leaf replica means creating a “leaf” with similar permeability of water and...

  27. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    Published
    Sept. 6, 2017, 1:01 p.m.
    ...physically joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are...fragment joining, but this is fine for its goal of creating a choice of standardized constructs and tools ... regions. Golden Gate Assembly also allows the creation of standardized DNA constructs, but its use of... expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments...well-designed fragment overlap sections, while still creating scarless plasmid inserts.  Want to Learn More...and eliminates the annealing step usually used to create a double-stranded oligo. Generating an sgRNA construct...directly transformed into E.coli for plating, screening and generation of sgRNA. Generation of sgRNA libraries...

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

    Type
    Blog Post
    Published
    July 27, 2017, 1:11 p.m.
    ...lower its aggregation tendency, increase its brightness, and decrease the amount of light required for...Fluorescent proteins have enabled scientists to pursue creative research avenues previously unavailable to them...applicants but Xin and Fatima stood out for their creativity in the use of fluorescent proteins and the potential...Fluorescent proteins can be applied in a long list of creative ways. In her graduate work in Michael Lin’s lab...by using photoswitchable fluorescent proteins to create enzymes that can be turned on and off with light...researchers will be able to use similar designs to create a wide variety of enzymes and proteins with inducible... to produce HMOs, sensor binding results in an increase in fluorescence of cells that actively produce...

  29. Addgene Welcomes John Doench to the Advisory Board

    Type
    Blog Post
    Published
    Aug. 13, 2015, 2:30 p.m.
    ... primary screen to knowing what are the real hits is so much shorter…. it’s a great screening technology...come here, I need to spend time here, this is an incredible place.” Doench has spent most of his research...researchers in a variety of fields develop RNAi screens to answer their own research questions. Starting... For his post-doc, Doench worked on using RNAi screens to learn more about cancer biology in Ed Harlow...developing CRISPR pooled libraries for use in genetic screens and has contributed much of his knowledge to the...was completely justified… it has been just an incredibly useful technology.” He goes on to explain that...become more efficient in terms of editing and screening) such that thousands of individual edits can be...

  30. Hot Plasmids: Spring 2025

    Type
    Blog Post
    Published
    June 3, 2025, 1:15 p.m.
    ...fusing miniTurboID with dCas9 to create TurboCas, the Shilatifard Lab created a very quick and efficient way...find in this post: Glutamate indicators with increased sensitivity and tailored deactivation rates Addgene's...targeting interneurons Glutamate indicators with increased sensitivity and tailored deactivation rates By...2025).In this latest work, the team designed and screened a large library of iGluSnFR3 mutants, identifying...Podgorski, K. (2025). Glutamate indicators with increased sensitivity and tailored deactivation rates. bioRxiv...puromycin resistance gene makes stable cell line creation a breeze. Modular by design: Key elements (promoter...protocols for viral generation and stable cell line creation (Figure 2). Looking for more? Explore our empty...

  31. Antibodies 101: Secondary Antibodies

    Type
    Blog Post
    Published
    Dec. 21, 2021, 2:15 p.m.
    ...this approach can greatly increase the sensitivity of an assay by increasing the number of signaling molecules...green; and proteins are represented in orange. Created with BioRender.com.    How do secondary antibodies...shorter than that of the primary antibody, due to increased binding affinity, many indirect antibody protocols...are designed to take place over multiple days. Increased run time is considered one of the major drawbacks...specific isotype subclasses of primary antibodies can increase the possible number of target proteins.   The ...immunohistochemistry advances have found ways to increase signals from conjugated primary antibodies. The...as nanobodies, has introduced new options for increasing signal strength.  For the time being, though,...

  32. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    Published
    April 6, 2017, 1:02 p.m.
    ... the untagged allele. Created with BioRender.com. The first step in creating the plasmids was to identify...be prohibitively expensive for many labs. We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously...first exon of the gene. In this manner, we have created an initial set of 10 plasmids representing 10 human...linker, and the GFP tag sequence to be inserted. Created with BioRender.com. A key feature of our methodology...the double strand break and introducing the tag. Created with BioRender.com. Plasmids, plasmids everywhere...strategy and design described above can be used to create similar donor plasmids for introducing tags into...

  33. Plasmids 101: Optimizing Plasmid Yields

    Type
    Blog Post
    Published
    May 26, 2016, 2:30 p.m.
    ... older plates could increase plasmid loss or mutation. Let it incubate Increasing the incubation time ...prep We'll discuss the following techniques to increase plasmid yield. Keep in mind that you might need...particularly stubborn prep. Check the bacterial strain Increase the volume Improve the culture media Let it incubate...more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture volume...you are working with a low copy plasmid. When increasing the culture volume, be aware that a higher volume...origins of replication (e.g. p15A, ColE1) can be increased by adding chloramphenicol to the culture medium...glycerol or glucose (in moderation) may also serve to increase cell density and plasmid yield. Start with a single...

  34. Editor's Choice, October 2016

    Type
    Blog Post
    Published
    Nov. 4, 2016, 2:30 p.m.
    ...Laws Bring Increased Postdoc Wages Our most popular blog post this month shares incredibly important information...enacted labor laws will likely increase postdoc earnings in the U.S. This increase, while overdue in my opinion...considering a postdoc. In “Changing Labor Laws Bring Increased Postdoc Wages,” Future of Research executive director...variety of downstream effects including possibly decreasing the number of postdocs various academic institutions...Science Communication Changing Labor Laws Bring Increased Postdoc Wages I hope you all have a great November...

  35. Great Results Start with Great Standard Curves

    Type
    Blog Post
    Published
    Aug. 15, 2024, 1:15 p.m.
    ...experiment. Here, we will provide an overview of how to create and use a standard curve and provide some general... I make the standards for a standard curve? You create a standard curve by serially diluting a known control...mix the samples well by vortexing or inversion. Created with BioRender.com.   When choosing a standard... vortexing between each step of the series. To increase the accuracy of the curve, avoid pipetting small...value. How do I plot and use a standard curve? To create a standard curve, you graph the measured response...wider span of concentrations but suffers from decreased specificity, especially as it approaches the minimum... all of the samples in the assay, as this will increase the accuracy of the data. For assays involving...

  36. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

    Type
    Blog Post
    Published
    Nov. 6, 2018, 1:12 p.m.
    ...transdifferentiated in vivo to pancreatic β-like-cells with the expression of pancreatic and duodenal homoeobox... [13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal tissue [17] Lung epithelial...rare in mammals, an example is observed in the pancreas when excess β-cell damage results in the transdifferentiation...cell types such as fibroblasts, hepatocytes, and pancreatic exocrine cells have been successfully transdifferentiated...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(4.... 38. Thorel, F., et al., Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-...PubMed PMID: 3690668. 40. Ferber, S., et al., Pancreatic and duodenal homeobox gene 1 induces expression...

  37. Hot Plasmids - November 2023

    Type
    Blog Post
    Published
    Nov. 14, 2023, 3:35 a.m.
    ...enable the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target ...reprogram delivery vehicles like lentiviral vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.... The Ting lab recently made two deposits that increase the utility of this technique.  By engineering... O’Neill The Cohen lab and colleagues recently created a system for recording the transcriptional history...resembling a “ticker tape” recording. The authors created externally-timed bands of color with HaloTag-iPAK4...thin sections of tissue, the Wu Lab is working to create a three-dimensional atlas of protein expression...-95 [K28/43R] (70 µm mounted sagittal section). Credit: Zhuhao Wu, Weill Cornell Medicine.  Interested...

  38. Data Freedom: The Expansion of Data Sharing in Research Publications

    Type
    Blog Post
    Published
    Aug. 5, 2014, 6:51 p.m.
    ...Jim Woodgett. Public Library of Science (PLOS) created a stir earlier this year when it announced its ...manuscript submission. The rationale includes increasing the ability of reviewers (and, once published...some cases, typically consortia involving genomic screens, the data stream is so prolific that raw information...require access to each individual video. To PLOS’s credit, the organization subsequently clarified what the...and this alone is a reasonable motivation for increasing both data accessibility and methodological detail...Minimally, raw but sufficiently annotated data used to create charts and tables in the paper are in scope. For... up new opportunities for research as well as increasing transparency. Thank you to our guest blogger...

  39. Modulate the Activity of 17 Signaling Pathways with One Kit!

    Type
    Blog Post
    Published
    March 22, 2016, 2:30 p.m.
    ... to each to make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...the only way to use the kit. “You could use it to screen for pathways that confer any phenotype of interest...drug resistance,” he said. “It could [be used to screen for] morphological features, migration, or survival...use our libraries to do things - sometimes drug screens and sometimes totally different experiments we ...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ... cancer screening...

  40. Five Popular Model Organisms

    Type
    Blog Post
    Published
    April 11, 2019, 12:39 p.m.
    ...developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, a method that...of quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling." Proceedings...these systems check out Gerald Rubin’s lab who created a set of modular vectors that can be used to optimally...unicellular organisms, they can also be used to easily screen multiple therapeutic compounds in one experiment...embryos with DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model...β-cells that originate in different regions of the pancreas and produce different levels of insulin (Hesselson...

  41. Filming Science Videos in the Age of Social Distancing

    Type
    Blog Post
    Published
    May 20, 2021, 1:15 p.m.
    ...Creating great educational science videos is tough. Is the content accurate? Are we conveying complex...people in tight locations. Conference rooms for on-screen presenters! Labs and benches! Everything was out...videos. It’s really nice to have a scientist on-screen, somebody who really knows the protocol and can...would be to use an iPad to shoot.  Build: Jennifer created a mobile camera area that included iPad and her...where I could focus on camera and audio, and the on-screen talent could just focus on their interview or performance...re-opened very early in the pandemic with some incredibly strict safety protocols to keep Addgenies safe...camera) would remain masked, it freed us up to be creative.  Test: Our first official shoot went off with...

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

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ...Symptoms of AIDS include rapid weight loss and an increased risk of infection, including both common infections...ART-treated patients lack plasma HIV-1, they are at increased risk for other chronic diseases, including dementia...disorders, nerve damage, and cardiac disease. This increased disease risk is attributed to latent HIV reservoirs... used histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. ... enhancer region near the NF-KB binding sites increased activation of HIV promoter-luciferase constructs...microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated HIV-1, ...cell. ZFNs and CRISPR/Cas9 have also been used to create mutations in genes essential to HIV-1 replication...

  43. SunTag and Fluorescent Imaging

    Type
    Blog Post
    Published
    March 28, 2017, 2:30 p.m.
    ...scaffold. To increase expression, Tanenbaum et al. modified the GCN4 sequence to increase its alpha-helical...activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing... Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named ...protein is expressed at low levels, you have to increase your imaging time to get enough signal. This workaround...cells without affecting protein function, thereby creating a single-molecule reporter of intracellular processes...alpha-helical structure and stability, creating v4 SunTag. Since the v4 system does not display protein aggregation...

  44. Understanding GPCR Signaling Using TRUPATH

    Type
    Blog Post
    Published
    Dec. 14, 2020, 2:15 p.m.
    ...resulting in decreased BRET2 signal. With increased concentration of drug, the BRET2 signal decreases. ...leads to downstream effects.  Bryan Roth’s lab created a collection of plasmids called TRUPATH to interrogate...the complex will disassociate, resulting in a decreased GFP2 emission. The activation state can be measured...showing the effectiveness of using TRUPATH as a screening technique. Lastly, the activity of both antagonists...plasmids are available as a collection, facilitating screening across the transducerome, to gain a more complete...

  45. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    Published
    June 2, 2020, 1:15 p.m.
    ... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring... attached fluorescent protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry...

  46. Multiplexed Overexpression of Regulatory Factors (MORF) collection

    Type
    Blog Post
    Published
    March 30, 2023, 1:15 p.m.
    ...finding. Transcriptional program changes from this screen successfully differentiated cells resembling all...from Joung et al.)    Putting MORF to work Screens and selections In its current format, the MORF ...library is most immediately useful for TF library screens. Previously engineered TF libraries were not as... library can be used with other selection and screening mechanisms, although the unique barcodes for each...mutation and/or TF dysregulation. A targeted TF screening approach can not only lead to a better understanding...better simulate a disease in a model system. TF screens can be designed to identify custom TF programs ...Cas-9 knockout and transcriptional activation screening. Nat Protoc. 2017 (4):828-863. doi: 10.1038/nprot...

  47. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    Published
    Feb. 28, 2023, 2:15 p.m.
    ... complexity of the device increases, as does the price. With this increased complexity comes the need ...adapted and combined with creativity and feedback from the community to create state-of-the-art devices...via workshops, seminars, or hackathons greatly increases accessibility. Through this approach, the Planktoscope...Madagascar. Platforms that allow for on-going creativity with the construction plans. Appropriate open-source...sources within the next few years, with the goal of creating more transparent and reproducible science in the...

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

    Type
    Blog Post
    Published
    Nov. 5, 2019, 1:28 p.m.
    ...approach, the Anna Cereseto Lab created pX-evoCas9, which they showed also increased specificity. Most recently...broader PAM specificity can target more sites. Created with BioRender.com. Specificity Specificity...multiple PAM sequences (NG, GAA, and GT), greatly increasing the number of genomic loci that can be targeted...developed SpCas9-HF1. Both of the enzymes showed decreased off-target activity relative to Cas9. Since then...accurate Cas9 (HypaCas9). This nuclease showed decreased or similar off-target activity, relative to eSpCas9..., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity." Nature communications 9.1 (2018...

  49. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    Published
    April 14, 2022, 1:15 p.m.
    ...new Antibody Guide and not just because I helped create it. But while antibodies, as the OG (as the kids...reagents we just didn't have the space to give proper credit to. Thankfully, as Addgene's resident blog editor...the majority of these alternative reagents are created in the lab, instead of generated in vivo, they ...Listed in no particular order.) Fab fragments are created through enzymatic cleavage of the variable region...in clinical applications. Fab fragments can be created in lab in situations where a suitable antibody ... fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. They are...cheaper to make than antibodies. Minibodies are created by binding together two scFvs with a CH3 linker...

  50. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    Published
    July 14, 2021, 1:15 p.m.
    ...antibodies produced from its serum several weeks later. Created with biorender.com. To generate polyclonal ...additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer is tested following...epitopes of the target protein. While this can increase background staining (see next section), it may...experimental protocol, using a polyclonal antibody increases the likelihood that one of the clones will still...crosslinking (ChIP) or fixation (IHC). Because of their increased sensitivity, polyclonal antibodies are particularly...epitopes of their target protein thereby greatly increasing signal amplification.  When to stay away from...population recognizes many different epitopes, increasing the likelihood of cross-reactivity with other...
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