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We narrowed to 784 results for: SCR;

Showing: 201 - 250 of 784 results

  1. Hot Plasmids - June 2019 - Optogenetics, Acoustic Reporter Genes, microRNAs, and the CRISPR-Cas9 system CHIME

    Type
    Blog Post
    ...recognized as important regulators of post-transcriptional gene expression across the majority of cellular...al., Nat Commun, 2019. PubMed PMID: 29934631. Screening immune cell lineages in vivo using the CRISPR-...tools have been used in immunology for in vitro screens of immune cell activity. However, a CRISPR-based...gene. CHIME can also be used for high throughput screens using pooled libraries. Find the CHIME plasmids...

  2. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    ...catalog – so check there before building one from scratch! Why deliver these systems virally instead of by... preserving cell viability. Library delivery Screening libraries, large collections of biological independent... entire genome, overexpression of all human transcription factors, barcoded libraries to trace cell lineage...library cargo delivered are controllable. For many screening applications, it is essential to deliver a single...

  3. Building and Validating Recombinant Antibodies: A Case Study

    Type
    Blog Post
    ...plasmid to express the target antigen could be screened in a high-throughput manner. Cells were labeled...which was observed for the hybridoma mAb.  While screening by IF-ICC worked well for identification of the...was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas and, ...limited the number of clones that needed to be screened through functional assays. Following functional...

  4. GPCRs: How Do They Work and How Do We Study Them?

    Type
    Blog Post
    ...parallel receptorome expression and screening via transcriptional output - TANGO) (Kroeze et al., 2015...fusion to a receptor-transcription factor fusion. TEV protease cleave the transcription factor from the receptor...

  5. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...which is removed by culturing cells at 38℃, but screening cells for the presence of transgenes is still ...Sendai virus doesn’t rely on promoters for transcriptional regulation, so it requires a different approach...in practice integration can occur. Okita et al describes a protocol for generating iPSCs by plasmid transfection...Methods RNA Delivery: For this approach, in vitro transcribed RNAs are serially transfected using a cationic...transduction, such as the polyarginine peptide described in Zhou et al and Kim et al. This is considered...

  6. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ...may not result in any cells, or all the colonies screened might not contain the correct plasmid. There’s...pick the big colony and you are safe! Colony screening Pick a few colonies (5-10) and grow them in a ... you've identified potential clones after the screening, sequence the plasmid. Many vectors contain sequences...can use the chromatogram to spot whether any discrepancies between your sequencing result and the expected...

  7. Plasmid Tools for Microbiome Studies

    Type
    Blog Post
    ..., the ability to detect mRNA transcripts of toxins and other transcripts of interest is a benefit of this...the organisms present while sequencing of all transcripts gives clues into what the microbes are doing....delivery into recipients by conjugation. The labs screened for the host-range of the plasmids and suitability...

  8. What's New in CRISPR - November 2021

    Type
    Blog Post
    ...read more content related to CRISPR technology, subscribe to our CRISPR blog content.   In this post we ...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...deletions and introduce epitope tags and RNA transcription start sites. The lab further demonstrated that...comprises an active Cas9 fused to the reverse transcriptase (RT), which induces two double-strand breaks...

  9. Announcing the Winners of the 2021 Michael Davidson and Roger Tsien Commemorative Conference Awards

    Type
    Blog Post
    ...indicators. Tian developed a video-based high-throughput screen for directed evolution of biosensors based on their... to engineer many types of biosensors.   This screening method allowed Tian to optimize a red‑shifted ... the improved voltage indicator coming from the screen to understand mouse cortical and hippocampal neural...neural microcircuits. “In the longer term, this screening platform may help us to create high-performance...

  10. Working with Nuclear Receptors

    Type
    Blog Post
    ...ligands NRs represent the largest family of transcription factors found in metazoans. The superfamily ...profiling datasets in to our data mining tool, Transcriptomine. We are currently nearing the completion of...transient transfection platform for assaying transcriptional activity, for example, was a key development...coregulators: Judges, juries and executioners of transcriptional regulation. Molecular Cell 27, 691-700. (PubMed...

  11. 5 Steps to Writing Grant Proposals for Grad Students

    Type
    Blog Post
    ...includes overview information, key dates, funding description, research plan, award information, eligibility...aspects are understood. While reading the funding description, pay attention to the keywords used and think...grant applications restrict the space available to describe the proposed work. Thus, every single sentence...project are clearly stated and align with the description of the RFA and funding institution. Emphasizing...

  12. Design Tips for Prime Editing

    Type
    Blog Post
    ...on a fusion enzyme of Cas9 nickase + reverse transcriptase and a prime editing guide RNA (pegRNA). The ...) complementary to the genome and a reverse transcriptase (RT) template including the desired edit. ...., 2019). Test different lengths of reverse transcriptase template, starting with about 10–16 nucleotides...pegRNA primer binding site (PBS) and reverse transcriptase template (RTT). Created with BioRender.com. ...

  13. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ... by T7 promoters to allow for bidirectional transcription to create dsRNA. Students can produce DNA templates...A recent publication [1] from the Sánchez lab describes the plasmids and protocols required for both WISH...the temporal and spatial localization of mRNA transcripts in tissue. This is useful for labeling specific... which are available through the deposit) are described in the Sánchez lab publication [1] so that the...

  14. Hot Plasmids and Viral Preps - January 2021

    Type
    Blog Post
    ... protein to date By Justin Ng High-throughput screening methods, such as fluorescence-activated cell sorting...s lab developed a new form of FACS-based cell screening, Single-cell Phenotypic Observation and Tagging...plasmids here Reduce the scale of your CRISPR screens by using this lentiviral CRISPR human genome-wide...guides per construct and allows a reduced scale in screens without any reduction in performance compared to...

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

    Type
    Blog Post
    ...loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a colorimetric readout that can... 45 minutes. The team deposited the reverse transcriptase, protease, and Bst polymerase constructs from...toolkit include ones for expressing MMLV reverse transcriptase and Taq polymerase that can be used in a one-step...Hsu PD (2021) Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors modulating...

  16. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    ...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc 12:828–863 . https:/...ask the depositing laboratory to review the discrepancies. If the depositing laboratory confirms that ... use a modified version of the Python program described by Joung et al. 2017, which we hope to make available...

  17. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...for viral titration consider using a reverse transcriptase mutant in a separate control reaction. This ...results. A packing vector deficient in reverse transcriptase will produce low levels of virus and can be ...into a target cell’s genome. This approach can discriminate multiple integration events, reducing the possibility... How Lentivirus is Used in CRISPR Genome-Wide Screening Learn How Lentivirus Has Been Used for Lineage...

  18. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...Maria were encouraged by previous reports that described magnetic responses of cryptochrome proteins or... and the team set up a directed evolution and screening experiment with three of the proteins that had...several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet variants were showing no... suggest she deprioritize the project, Maria’s screens of AsLOV2 variants showed some significant gains...

  19. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    ...up, most will eventually grow within a week. We screen the wells in groups as they become confluent, taking...important to have a functional assay in place to screen your clones. Depending on the protein, we have ...SH-SY5Y cells, as these cells did not seem to form discrete colonies but constantly migrated away from each...either initially, or using secondary biological screens. For example, for cancer researchers, if growth...

  20. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...obtained if any single component was omitted. Screening for successful Golden Gate Assembly All Golden...to faster assembly workflows that require less screening to confirm the accuracy of assembly. The fidelity... assemblies per plate using outgrowth volumes described in Figure 3, with calculated yields from entire...

  21. Transferable Skills: Negotiation

    Type
    Blog Post
    ...how much data is necessary before submitting a manuscript. In fact, how many of you have negotiated over...over the order of authorship for a manuscript? And that’s only in your scientific life! I’m certain that...mate would have time to make comments on your manuscript. If you give her this slot, she’ll have extra...her deadline) to help you. Her feedback on your manuscript is more important to you than collecting your...

  22. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    ...proteins involved, with the goal of being able to discriminately and effectively eliminate cancerous cells from...lines. Further, for each plasmid in the kit, the transcript most commonly found across all TCGA tumor samples...he or she can use to study it (as seen in the screenshot below). Currently, the page includes interactive... Tackling Cancers' Drug Resistance with a New Screening Kit Learn How the Clon Tracer Library Can Be Used...

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

    Type
    Blog Post
    ...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label ...world examples of these situations in cells are described below (See Figure 1).   Figure 1: Each... not necessarily monomeric, even if they were described as monomeric in a publication. The gold standard... [2]. Some affinity assays used in the papers describing the FPs above including molecular sizing columns...

  24. Tips for Using BLAST to Verify Plasmids

    Type
    Blog Post
    .... At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as mismatches, deletions...determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs are optimized...matrix BLOSUM62, but you may want to check the description of the other matrices to see if another would...plasmids instead! All BLAST images are modified screen shots from the NCBI BLAST website. Additional Resources...

  25. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    ...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note...

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

    Type
    Blog Post
    ...type of interest. Repeat rounds of infection and screening by PCR enrich for capsid variants with increased...of Chan et al, was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly...brain. Delivering the CREATE AAV library via IV screens for capsids that are able to cross this barrier...AAV as a tool for gene expression in mammals. Subscribe to Viral Vector Blog Posts Additional Resources...

  27. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ...Cas9 expression     The requirement for Cas9 transcription and translation delays editing What are Cas9...from Alex Schier’s Lab. gRNAs can be in vitro transcribed from ssDNA, which can be generated by commercial...targeted approach using RNPs has recently been described in vitro, with the hope of eventually using this...PMC5681539. Additional resources on the Addgene Blog Subscribe to CRISPR blog posts Learn about other CRISPR ...

  28. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

    Type
    Blog Post
    ...an efficient gene activator when fused to a transcriptional activation domain (Karmakar et al., 2024). ... using your method of choice. Step 5: Colony Screening Use Primer 92F (5′-cattacgcaattggacgacaac-3′)...′) and Oligo 2 (specific to your target) to screen colonies via PCR. A 354-bp product confirms successful...insertion. Alternatively, use M13R and Oligo 2 to screen. A 667-bp product confirms successful insertion...

  29. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    ...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants that are still...deactivation tools for zebrafish Random mutagenesis screens provided a major toehold into understanding how...one-eyed pinhead mutant from the random mutagenesis screens. Microscopy-based lineage tracing can help illuminate...

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

    Type
    Blog Post
    ...policy and recently announced that of the 16,000 manuscripts that had been processed since the declaration...access of this data is to be made at the time of manuscript submission. The rationale includes increasing...some cases, typically consortia involving genomic screens, the data stream is so prolific that raw information...issue by requiring ever more condensed methods descriptions, legends and limiting numbers of figures such...

  31. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ...processing (Figure 1). The shRNA sequence is transcribed to produce hairpin shRNA structures that are ...fact! Endogenous miRNA production starts with transcription of the miRNA to create pri-miRNA, which is then...typically use RNA polymerase III promoters, which transcribe small RNAs. The most common promoters are U6,... H., & Kim, V. N. (2004). MicroRNA genes are transcribed by RNA polymerase II. The EMBO Journal, 23(20...

  32. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ... editor or adenine base editor) and reverse transcriptase-mediated prime editing technologies have been... through Addgene. In addition to the plasmids described above, Addgene carries several useful CRISPR toolkits...Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiol 169:971–985 . https...CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922. PLoS ONE 11:e0154027 ....

  33. Evolution of Lab Techniques

    Type
    Blog Post
    ...including easier automation, higher throughput screening, and a lower requirement for the amount of DNA...transgenic animals may choose Southern blotting to screen founder transgenic animals for the inserted transgene...below), restriction mapping may still be used to screen multiple colonies before selecting a smaller subset...prevails. Sequencing whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples...

  34. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    ...perform in vitro transcription, and use the IVT product as a template for reverse transcription to create your...process of mouse model generation simpler, as described in an earlier post on generating CRISPR mouse ...'d use ivTRT to create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids...

  35. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...these cases, screening multiple bacterial colonies using one of the detection methods described above can...confirmations like linear and nicked plasmids, as described in this article.   Long-read sequencing Addgene...by growing seed stocks at 30°C compared to 42°C. Screen multiple colonies to isolate the monomer Sometimes...

  36. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...inserted in this LTR so you don’t need Tat to transcribe the viral genome in producing cells. Currently...process is to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the...transduced by a GFP encoded virus, and the reverse transcriptase activity assay uses qPCR to quantify viral RNA... RNA. All of these methods have been described in a previous Addgene blog Once you know your viral titre...

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

    Type
    Blog Post
    ... to screen for positive clones in most cases, and restriction enzyme digest can be used to screen plasmids...BAC DNA can be modified with any of the below described approaches.    Bacterial strain with integrated...recombination than dsDNA substrates, so you should screen more clones when using dsDNA. Have you used the...

  38. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ... been described in this blog. If your data does not meet the assumptions for the tests described above...to not have any effect on each other, they have discrete effects on the dependent variable, and those variables...error rate than the multiple comparison tests described above. Violated assumptions and statistics Statistical...

  39. CRISPR Guide Update

    Type
    Blog Post
    ...update, we have added new sections to share and describe some incredible new CRISPR technologies developed...much-anticipated prime editing section and additions describing CRISPR’s ability to make large genome edits using...transposases or the Cascade-Cas3 system. We also describe newly engineered Cas9s with more flexible requirements...

  40. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...levels to generate anything from individual transcription units (TUs) used to express your favorite gene-of-interest...genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more....can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these...are designed for flexibility. The Fragmid kit described above works in both mammalian systems and Drosophila...

  42. Addgene's Website Update

    Type
    Blog Post
    ... The new search bar.   Pro tip! You can use a manuscript title to search for plasmids from a specific ...quickly navigate around the Addgene website without scrolling up and down the page.  Figure 3: The Education... Tools menu Further down What happens if you do scroll down? You’ll find Blugene, as well as information...

  43. Fluorescent Proteins 101: History of Fluorescent Proteins

    Type
    Blog Post
    ...characteristic radiance for some time – there are records describing them dating back to the first century A.D. – ...glowing protein could be used to measure gene transcription, as well as to track protein localization. And...mechanistic understanding of GFP’s function. His lab described the protein’s structure and also discovered many...

  44. Designer PUF Proteins for Any RNA Target

    Type
    Blog Post
    ...ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become...proteins The tool she and her colleagues led by Zhao described and demonstrated in a report published in The ...of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA. J Biol Eng. 2014...

  45. Editor's Choice, September 2016

    Type
    Blog Post
    ..., P1 phage. 2) The post describes an ingenious but simple technique to screen for proteins that could ...Jessica Sacher from the University of Alberta and describes a technique you can use to find phage proteins...

  46. SciComm with the Experts at Science in the News Part 2

    Type
    Blog Post
    ... discusses some of the programs SITN runs and describes some of the more popular topics from the Science...telling. Chapter 5: The SITN Blog - Vini and Amy describe how the SITN blog is a source of scientific information...underwater? Chapter 8: Work-Life Balance - Vini describes how she balances working in the lab and working...

  47. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    ... paired DSBs. This method allows for deletion screening by conventional PCR. Biallelically disrupted cells...transfected cells via FACS, primer validation, and screening by PCR for biallelic deletion clones:    ...video? The Orkin and Bauer lab's related paper describes their methods for CRISPR design, cloning, and ...

  48. Tag Your Favorite Yeast Genes with Ease

    Type
    Blog Post
    ...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

  49. Working 9-5: an Addgene Co-op

    Type
    Blog Post
    ...process When I first saw the comprehensive job description for this co-op, I was immediately interested ...had stood out to me because it gave me a full description of the tasks I would be responsible for, in comparison...and Geneious, I was able to determine if any discrepancies between what we sequenced in house and the submitted...

  50. Year of the Camelid: Antibody Style

    Type
    Blog Post
    ...camelids too, though not just for the reasons the UN describes. In honor of the Camelid family, I wanted to take...of times on this blog over the years and have described how they compare to other antibodies and affinity...sdAbs were used to create GFP scaffolds for transcriptional activation.  sdAbs in the clinic sdAbs have...
Showing: 201 - 250 of 784 results