We narrowed to 612 results for: SCR
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TypeBlog PostPublishedFeb. 10, 2015, 3:06 p.m....specialized strains are available to confer greater transcriptional control, assist with proper protein folding...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...addition of IPTG. T7 RNAP is then available to transcribe the gene of interest from a T7 promoter on the... the expression plasmid in order to repress transcription from a hybrid promoter. Although the DE3/T7 ...don't see protein overexpression? The strains described above should generate sufficient expression levels...
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Filming Science Videos in the Age of Social Distancing
TypeBlog PostPublishedMay 20, 2021, 1:15 p.m....Jennifer and I spent about an hour that morning scrambling. Everything we had set up involved multiple people...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...together a video from their recorded answers. No scripting made this a lot easier and I didn’t need to be...where I could focus on camera and audio, and the on-screen talent could just focus on their interview or performance... -
Multiple Plasmids at a Low Price: Kits from Addgene
TypeBlog PostPublishedSept. 14, 2016, 2:30 p.m....genes from a single transcript, or if you want to prevent read-thru transcription from resulting in the...3509-3514. Tikhonova, Elena B., et al. "A transcription blocker isolated from a designed repeat protein...protein combinatorial library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid ...the production of a downstream peptide, the transcription terminators in this series are just the tool... -
Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?
TypeBlog PostPublishedMay 10, 2016, 2:30 p.m....therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using ...histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. For a targeted...dCas9-VP64 fusion commonly used to activate transcription, but with two additional activation domains ...domains (MS2 and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target ...to Use CRISPR Pooled Libraries for Genome-Wide Screens Additional Resources onf Addgene.org Check ... -
Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons
TypeBlog PostPublishedApril 24, 2015, 2:39 p.m....R26R-Confetti described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine...overcoming the limitations listed above. First, they screened a variety of fluorescent proteins to find those... via immunostaining. This feature facilitates screening of Cre-negative Brainbow mice to determine the...addition of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), commonly used to... -
Addgene Protocols Go Viral: AAV Purification and Titration
TypeBlog PostPublishedJune 13, 2019, 12:19 p.m....article describing a sucrose supplementation strategy to improve yield. In addition, as described in this...you time and improve your experiment results! Subscribe to the Addgene YouTube page! AAV purification ...lab tips, and more video content from Addgene, subscribe to Addgene’s YouTube Channel. Additional resources... -
What's New in CRISPR - Winter 2018
TypeBlog PostPublishedDec. 18, 2018, 1:17 p.m....Find the anti-CRISPR plasmids Tagging human transcription factors with GFP Kevin White’s lab recently ...deposited a set of CRISPR plasmids to tag human transcription factors with GFP. As part of the Encyclopedia...these donor plasmids will help researchers map transcription factor localization and binding via ChiP-seq...ChiP-seq. Learn more about ENCODE Find the transcription factor tagging plasmids If you have a new CRISPR... -
Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation
TypeBlog PostPublishedMay 21, 2020, 1:15 p.m....tools in Drosophila (Fig. 1): GAL4-UAS, a transcription factor binding site. GAL4 expressed in the fly...site to cleave and inactivate any read-through transcripts of the effector gene. Effector genes, any gene...across cell types and organisms by replacing the transcription factor binding site and effector in the construct... the SPARC system to use other experimental transcription activation systems, such as LexA/p65 or mCD8... -
CRISPR 101: Drug discovery and CRISPR
TypeBlog PostPublishedMay 4, 2023, 1 p.m....CRISPR technology has brought drug and genetic screens together and expedited the drug discovery pipeline...Alternatively, a disease model can be generated, and drugs screened for a change in pathology. If the disease pathology...accelerate the drug pipeline. High-throughput CRISPR screens can be used to inhibit, activate, or knockout many...identified several candidate inhibitors of gene X in a screen. You generate a gene X knockout in a cancerous ... -
Five Questions to Ask If You're Deciding Between a Master's or PhD Program
TypeBlog PostPublishedFeb. 16, 2021, 2:15 p.m....a job search website like Indeed. Do the job descriptions sound interesting? Is the work something you... types of jobs people with master's have Job descriptions often fail to capture all of the responsibilities...responsibilities of a role, so it helps to see how people describe their actual work. To do this, try looking at ...to present your work at conferences, help with manuscript and grant preparation, and lead your own research... -
Walking beside Academia and giving it a high five: My transition from graduate work to a position at Addgene
TypeBlog PostPublishedJuly 2, 2015, 8:37 p.m....Was I jaded by my time in academia? Did I run screaming from the lab like the cartoon on the right?!? ... course more quickly than writing scientific manuscripts and, as one of my supervisors warned me, the ...’t have to mean abandoning it or running away screaming, but instead can involve walking alongside it ...it and giving it a high five. While screaming can be therapeutic and I’ve certainly done my fair share ... -
Addgene @ Keystone: Thoughts on Precision Genome Engineering and Synbio
TypeBlog PostPublishedJan. 15, 2015, 1:50 p.m....fly fishing, and local grizzly bear attacks. He described one such recent attack as "hilarious". Welcome... Mehmet Fatih Bolukbasi from Scot Wolfe's lab described a chimeric Cas9 that is improving the nuclease...also got to show off our new CRISPR page (which describes how to plan your CRISPR experiment for our new...next week, so follow us on Facebook, Twitter, or subscribe to this blog. Find more CRISPR and Genome Engineering... -
Antibody Validation for Flow Cytometry
TypeBlog PostPublishedSept. 10, 2024, 1:15 p.m....see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity...of antibody validation, and it is recommended to screen multiple candidate antibodies when possible. If...commercially available. Various proteomic and transcriptomic datasets can be used to select a candidate ...recognized by either antibody was not shared. Tables describe that the antibodies both recognized the target...experiment. We find it useful to start with a screening procedure in easily genetically modified cell ... -
Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS
TypeBlog PostPublishedJuly 21, 2017, 12:48 p.m....more detailed level, the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It...workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences...sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus...Lai, T. Lee, Genetic mosaic with dual binary transcriptional systems in Drosophila., Nat. Neurosci. 9, 703... -
Unique and persistent IDs for improved reproducibility: Addgene now supports RRIDs and compact identifiers for all plasmids
TypeBlog PostPublishedNov. 20, 2018, 8:17 p.m.... lacks context unless it links to a verified description of that reagent online. Furthermore, if the link... link between the catalog number and reagent description is lost (e.g. a supplier is bought out by another...specific information about the plasmid: name, description, how the plasmid was generated, validated sequence...our example plasmid, lenti-Cas9-VQR-Blast. If you scroll down to the bottom of this plasmid’s page you will... -
With Our New Viral Service, We're Taking CRISPR Further!
TypeBlog PostPublishedDec. 6, 2016, 3:30 p.m....CRISPR pooled gRNA libraries that can be used to screen for genes involved in a wide variety of cellular... infect target cells, and, finally, conduct the screen. To help alleviate some of this labor for our ...optimization-scale infection and at least one screening-scale experiment achieving the gRNA representation...) says: "When you're setting up to do a genetic screen, you need to focus on building a really good model... -
Components of CRISPR/Cas9
TypeBlog PostPublishedFeb. 2, 2016, 5 p.m.... of the major CRISPR lingo and will be able to describe the functions of the various CRISPR/Cas9 components...Bind to a guide RNA The guide RNA, which will be described in more detail below, enables Cas9 to cut a specific...arrays as found in the bacterial genomes are transcribed into pre-crRNAs containing both the spacer region...RNase III, the tracrRNA and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound... -
Synthetic Photobiology: Optogenetics for E. coli
TypeBlog PostPublishedSept. 8, 2015, 2:30 p.m....responses are varied; one outcome is directed transcription from a given promoter. To harness the power ...the RR (OmpR) via phosphorylation, promoting transcription (see figure below). Subsequent exposure to red...response regulator via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red...types of proteins, including Cas9 and dCas9-transcription factors, for which low expression levels are... -
Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies
TypeBlog PostPublishedJuly 28, 2017, 1:30 p.m....Harvard University. In my previous two posts, I’ve described the fundamentals of how to work with Drosophila...Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study...red. The variation in color depends on how transcriptionally active the genomic locus of integration is...Using a Drosophila cell line, I’ve studied a transcription factor important for T cell differentiation ... -
Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity
TypeBlog PostPublishedNov. 5, 2019, 1:28 p.m....Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated from different..., and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which...Nishimasu et al., 2018). The David Liu Lab similarly described xCas9, which displayed the ability to target multiple...post on choosing a Cas9, the Feng Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed... -
Hot Plasmids - June 2019 - Optogenetics, Acoustic Reporter Genes, microRNAs, and the CRISPR-Cas9 system CHIME
TypeBlog PostPublishedJune 6, 2019, 12:41 p.m....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... -
Hot Plasmids December 2018 - C. elegans RNAi, TurboID, Detecting Viral Outbreaks, and Protein Binding Specificity Tools
TypeBlog PostPublishedDec. 6, 2018, 1:34 p.m..... elegans. These dsRNAs target specific mRNA transcripts for degradation. Despite how easy these experiments...target gene between two inverted T7 promoters. Transcription from each promoter progresses past the specific... Lab’s recent Nature Biotechnology paper, they describe TurboID and miniTurbo, which utilize non-toxic...include protein stability, small molecule binding screens, peptide arrays, and phage display selections. ... -
Building and Validating Recombinant Antibodies: A Case Study
TypeBlog PostPublishedApril 5, 2022, 1:20 p.m....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... -
Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems
TypeBlog PostPublishedMay 19, 2020, 1:15 p.m....sequencing by NGS. Addgene’s QC scientists immediately screen these plasmids to confirm they contain the expected...of plates will have select samples prepared and screened by NGS to spot check the samples, the rest will...are requested by scientists, will immediately be screened by our QC scientists to confirm the plasmids contain...By leveraging our core strengths of intensive screening and the use of high throughput platforms, Addgene... -
Viral Vectors 101: Viral Applications
TypeBlog PostPublishedApril 13, 2023, 1:15 p.m....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... -
GPCRs: How Do They Work and How Do We Study Them?
TypeBlog PostPublishedFeb. 11, 2021, 2:15 p.m....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... -
Delivery Methods for Generating iPSCs
TypeBlog PostPublishedApril 17, 2018, 1:37 p.m....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... -
Troubleshooting Your Plasmid Cloning Experiment
TypeBlog PostPublishedSept. 24, 2019, 12:55 p.m....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... -
Plasmid Tools for Microbiome Studies
TypeBlog PostPublishedJune 27, 2019, 12:25 p.m...., 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... -
What's New in CRISPR - November 2021
TypeBlog PostPublishedNov. 18, 2021, 2:15 p.m....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... -
Announcing the Winners of the 2021 Michael Davidson and Roger Tsien Commemorative Conference Awards
TypeBlog PostPublishedAug. 5, 2021, 12:15 p.m....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... -
Working with Nuclear Receptors
TypeBlog PostPublishedJuly 15, 2014, 4:03 p.m....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... -
5 Steps to Writing Grant Proposals for Grad Students
TypeBlog PostPublishedFeb. 11, 2014, 12:13 p.m....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... -
Educational Planarian Tools for Teaching Developmental Biology Techniques
TypeBlog PostPublishedMay 24, 2018, 1:20 p.m.... 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... -
A Look Back at One Year of Plasmid Sharing for COVID-19 Research
TypeBlog PostPublishedJuly 12, 2021, 1:15 p.m....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... -
Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene
TypeBlog PostPublishedApril 6, 2021, 12:30 p.m....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... -
Hot Plasmids and Viral Preps - January 2021
TypeBlog PostPublishedJan. 5, 2021, 2:15 p.m.... 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... -
Design Tips for Prime Editing
TypeBlog PostPublishedJan. 23, 2025, 2:15 p.m....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. ... -
Tips for Titering Your Lentiviral Preps
TypeBlog PostPublishedMarch 15, 2016, 2:30 p.m....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... -
Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity
TypeBlog PostPublishedOct. 11, 2018, 12:30 p.m....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... -
Cloning Mammalian Cells with the Agarose Method
TypeBlog PostPublishedSept. 7, 2017, 12:17 p.m....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... -
Transferable Skills: Negotiation
TypeBlog PostPublishedMarch 3, 2022, 2 p.m....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... -
Magnetic Control of Proteins: More than a Dream
TypeBlog PostPublishedSept. 24, 2024, 1:15 p.m....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... -
Addgene's Cancer Collection Pages Connect the Research Community
TypeBlog PostPublishedSept. 6, 2016, 2:30 p.m....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... -
When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells
TypeBlog PostPublishedApril 19, 2016, 2:30 p.m....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... -
Tips for Using BLAST to Verify Plasmids
TypeBlog PostPublishedMay 29, 2014, 1:29 p.m..... 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... -
CRISPR 101: Ribonucleoprotein (RNP) Delivery
TypeBlog PostPublishedSept. 6, 2018, 12:02 p.m....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 ... -
AAVs CREATed for Gene Delivery to the CNS and PNS
TypeBlog PostPublishedSept. 28, 2017, 2:01 p.m....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... -
CRISPR 101: RNA Editing with Cas13
TypeBlog PostPublishedJuly 31, 2020, 12:30 p.m....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... -
Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More
TypeBlog PostPublishedAug. 13, 2019, 12:58 p.m....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...