Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

In Category

We narrowed to 881 results for: NSI

Showing: 451 - 500 of 881 results

  1. Pooled Library Amplifications

    Type
    Blog Post
    ...and how will you ensure this?). You should also consider if there are any features of the library backbone...difficult to transform.    Figure 1: Factors to consider when creating a pooled library amplification protocol...BioRender.com.   The most important factor to consider is your desired library coverage, since this will...

  2. PhD Applications After COVID

    Type
    Blog Post
    ...in-person events change seemingly daily (and are inconsistent between universities!) This guide tells you ...to be incredibly useful, as it gives a more comprehensive idea of that advisor’s research interests which...program and faculty. Here are some questions to consider for your online meetings:  Is this department...

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

    Type
    Blog Post
    ...result in appropriate levels of colony plating densities. Ligase fidelity and assembly efficiency Five...possibility. Recently, NEB researchers profiled the comprehensive fidelity of cohesive end ligation by this enzyme..."A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias...

  4. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...carry out a high-throughput genetic screen, it is expensive and time consuming to individually generate gRNAs...Most insertions can only be detected by labor-intensive processes like PCR or by evaluating visual phenotypes...PMID: 27052166. Kunzelmann, Stefan, et al. “A Comprehensive Toolbox for Genome Editing in Cultured Drosophila...

  5. DNA Purification Without a Kit

    Type
    Blog Post
    ...you reach for that silica spin column, stop to consider some ways to purify DNA without a kit. DNA purification...advantages: they are convenient and provide uniform, consistent results. But they are also less accessible due... faster, cheaper, and had a similar level of sensitivity. Figure 2: Steps to purify DNA using a ...

  6. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ...reporter enzyme. For multicolor staining, careful consideration must be used when selecting which antibodies...conjugates are used, but these are usually more expensive than unconjugated primary antibodies. Reagent...subclasses of 1° for multi-color staining Can be expensive Nanobodies   Recombinant; do not require ...

  7. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    ...of looking for The One Method to Rule Them All, consider the following four questions to figure out which...than it is to design one demonstrating mitosis.   Consider also the amount and type of information you want...competitive while digital can be oversaturated. Transition from academic to informal writing can difficult...

  8. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    ...So… why do some people still use amp? Carb is expensive (relative to amp), and for some applications, ...with Amp, timesaving for transformations. Con: Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside...Like carb, spectinomycin is the more stable and expensive antibiotic compared to streptomycin. Spectinomycin...

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

    Type
    Blog Post
    ...luck, and always keep going. Appendix DNAse Hypersensitive Sites and Intergenic CRISPRs Yours truly generated... In reality, cellular DNA is a complex three-dimensional globule of chromatin. Chromatin is a combination...bowtie2 -f -x HG19_GENOME --local -f -k 10 --very-sensitive-local -L 9 -N 1 -U GRNA_23MERS -S GRNA_HITS.sam...

  10. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ...inhibit cell cycle regulatory proteins responsible for the transition out of S phase, so cells stay in an...exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful HDR ...

  11. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...present even in common lab cloning strains. This intrinsic bacterial recombination pathway, referred to as...are due to incorrect primer sequences! Primers consist of two regions: the 3’ end binds template DNA for... blog posts Read our blog post on 5 factors to consider before choosing a cloning method Check out all...

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

    Type
    Blog Post
    ...capsids were packed with a GFP transgene and GFP intensity and transduction efficiency of neurons were measured...AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains they were derived from...Additional Resources on the Addgene Blog Important considerations when using AAVs Learn more about using AAV ...

  13. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

    Type
    Blog Post
    ...Fluorescent Protein? GFP is a ~27 kDa protein consisting of 238 amino acids derived from the crystal jellyfish...point mutation that increased the fluorescence intensity and photostability of GFP. This also shifted its... blog, you should! He's got great articles and insight on the newest tools and techniques in fluorescent...

  14. Binning Singletons: Tackling Conference Networking When You Don’t Know Anyone

    Type
    Blog Post
    ... bins together at a mixer. We call this Quorum Sensing, after the phenomenon in bacteria that occurs when...gene expression in response to changes in cell density. At the mixer, they meet other Mentors and Singletons...briannadoesbio) June 22, 2019 Favorable conditions - Expansion of networks The last stage is Exponential Growth...

  16. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...exception) necessitates such molecular gymnastics for sensitive applications such as CRISPR screening. The plasmids...are often many more genetic strategies) and then consider whether traditional cDNA overexpression is suited...eBook Resources on Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources...

  17. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    ...?" is an open-ended list of proteins. Instead, consider "how do proteins X and Y regulate the Z pathway...that is easily visible from the back of the room. Consider going beyond the whiteboard. You may wish to create...blood sugar. Find an energy bar that you like and consider eating one before your chalk talk. Bring your ...

  18. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation...also constructed smaller vectors suitable for transient assays using protoplast transformation. Cytosine...72 hours post-transformation/transfection in a transient assay. For stable transformation, regenerated ...

  19. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...When choosing beads, there are several factors to consider. Agarose beads are sponge-like and vary in shape...express the protein of interest, such as a sample transiently transfected or stably infected with the protein...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect...

  20. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    ... the virus and is less affected.  Altering sensitivity to serum. Different pseudotyped viruses are differentially... protein that binds phosphatidylserine and low-density lipoprotein receptors (LDLRs) on a cell surface...CD, Friedman T, McClure MO, Weiss RA (1997) Sensitization of rhabdo-, lenti-, and spumaviruses to human...

  21. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ...pTargetF system consists of one plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon...changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection...

  22. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ... can be to get a “bad batch”); Customizable sensitivity and detection - since they frequently undergo...fluorescence for readout may render this approach less sensitive in most applications. Learn About Fluorescent ... sensor could save precious minutes in a time sensitive detection assay. In 1999, Grate and Wilson paired...

  23. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...clone. You will also need to use a E. coli strain sensitive to CcdB (e.g. DH5α, TOP10, Mach1). The ccdB gene... the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain ...sequences, start codon, stop codons, reading frame considerations, etc). This video demonstrates how to use the...

  24. Viral Production at Addgene

    Type
    Blog Post
    ...staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed. The...preparation shows that the vast majority of the vectors consist of full particles (white arrowhead) relative to...targeting integrated copies of the lentiviral Rev responsive element (RRE) gene and the cellular ribonuclease...

  25. 5 Reasons to Use Reddit for Science Communication

    Type
    Blog Post
    ...place and give you some reasons why you should consider using Reddit to share your science. While Reddit...permanent reference by the Winnower. The only small downside to the AMA from my end as an organizer/promoter... general advice on how to meet people, how to transition out of academia, and other science career topics...

  27. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    ...important to note that in Morgens et al., guides were considered toxic if they were depleted from the screen. ...sites that lack open chromatin marks, DNase hypersensitivity, or are in an enhancer, transcription factor... significant difference in depletion of ricin sensitizer genes identified with short and long guides. ...

  28. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ...vectors are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used...disadvantages to the AAV-based approach that must be considered when deciding whether to use AAV or CRISPR for... is a Professor of Oncology at the Lombardi Comprehensive Cancer Center, Georgetown School of Medicine...

  29. Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

    Type
    Blog Post
    ... to solve the problematically low fluorescent intensity of previous versions of Brainbow. Developed from...this system useful for visualization of cellular extensions, such as neurons in the fly visual system, which...cutaneous melanoma induced by mutant BRaf arises from expansion and dedifferentiation of mature pigmented melanocytes...

  30. March for Science

    Type
    Blog Post
    ...States government? Politics and science are intrinsically linked: governments exist in reality and science...include science in decision making, it will be considered a success. Below are resources for the march ... “politicized science” ABC News: White House considering gutting 38 EPA programs: draft budget  Science...

  31. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ...retroviral backbones. On some occasions, shRNAs are transiently expressed using either AAV backbones or species-specific...synthetic, mature siRNA. siRNAs are delivered via transient transfections and are handy for quick in vitro...time you need to knock down a gene, maybe you’ll consider using RNAi! References and Resources References...

  32. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ...material, which ensures the editor will only be transiently present in transduced cells, limiting off-target.... (2024). Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes...CRISPRoff system — a programmable epigenetic editor consisting of dCas9 fused to a DNA methyltransferase — that...

  33. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ... social science to an audience that I thought consisted of all biologists. The social scientists in the...a solution or overcame obstacles. Take time to consider the order of your narrative. Scientists do this...are worried that this might sound condescending, consider explaining the term on a slide but not reading...

  34. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    ...donor (e.g., sister chromatid, plasmid, etc.), and consists of three pathways: classical double-strand break...you need to generate a knock-in.   General considerations for designing a repair template to create mutations...cells than their plasmid counterparts. Another consideration for dsDNA templates is their ability to randomly...

  35. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    ...to be examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a ...determined in vitro by measuring the fluorescence intensity of purified protein at different acid concentrations... its fluorescence and therefore GFP cannot be downsized by deleting residues. Only about 10 amino acids...

  36. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...repetitive sequences, but if present, a minor extension can usually ensure that the 3’-base(s) do not ...manufacturers’ PCR protocol. Be sure to adjust the extension time to match the size of your template. Amplification...PubMed PMID: 2448875. 4. Xia, Yongzhen, et al. "New insights into the QuikChangeTM process guide the use of...

  38. Finding Your Perfect Job After University

    Type
    Blog Post
    ...industry positions. There were, however, some downsides to working in a large commercial company. In my...sometimes go unnoticed at large organizations. Another downside to large commercial companies which are reliant... treatment outcomes. I would say that the only downside of modern genetics from the perspective of somebody...

  39. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any protruding...below the primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending... instructions for specific instructions about extension time and temperatures. Initial Denaturation for...°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. Run 2 μL on a gel to check...The rate of DNA synthesis is ~1-2 kb/min. The extension time can be adjusted according to the length of... temperature around 70°C and is the molecule responsible for driving the DNA synthesis. Sterile dH 2 O...

  41. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...professional training after earning a doctorate Considered a "stepping stone" position to become a professor... lab Undergraduate student Roles and responsibilities can vary "Lab assistants" usually wash glassware...1-2 years) Often a gap-year position taken to transition to the next career stage (ex: grad school, med...

  42. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...the surface or the inside of your cells. While proteins themselves can emit intrinsic fluorescence when...fixable dye stains that react with free amines on and inside the cells and are thus typically used for distinguishing...

  43. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    ...sequences can be optimized for specificity and sensitivity, for example by changing certain amino acids ... that can pass through each well - the optical density (OD) - of the known and experimental wells is measured...is used to detect proteins on the surface of or inside whole cells. Antibodies - typically, primary antibodies...

  44. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...culture of bacteria transformed with your plasmid Resuspension buffer Denaturing solution Renaturing solution... This step gets all of the bacteria back into suspension, but within a smaller volume of buffer that is...Prepare the following solutions: Solution I - Resuspension Buffer 25 mM Tris-HCl (pH 8) 50 mM glucose 10...phase Middle phase - A white layer may appear, consisting of precipitated protein particles Bottom phase... mM Tris-HCl pH 8, 0.1 mM EDTA). Pro-Tip DNA resuspension can take time, it is a good idea to let it sit...eliminate RNA contamination. This is included in the resuspension buffer of most kits. Reference Page Top Index...

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

    Type
    Blog Post
    ...developed in David Liu’s lab in 2019, has added considerably more precision and flexibility to the CRISPR...engages the prime editor protein. The prime editor consists of a Cas9 nickase fused to a reverse transcriptase...mismatch repair   ✓   ✓ ~ ~ ~ Transiently co-express mismatch repair inhibitor MLH1dn ...

  47. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...monoclonal line could be generated at any time. Considerations Before You Start Some cell lines tend not to...optimized for adherent cells but can be modified for suspension cells. The number of monoclonal lines generated... this, you are seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5 cells...Figure 1: Generation of monoclonal cell lines from expansion of individual A549 cells stably expressing Cas9... in individual wells of a 96-well plate after expansion of single cells for 13 days. Note that growth ...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...

  48. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers...need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at...specific protocol and conduct an OD600 to measure the density of your culture if needed. A good negative control...culture. More aeration may help to increase the density of the culture. Normally cultures shake at 150 ... this to 350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

  49. Fluorescence Titering Assay

    Type
    Protocol
    ...applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell... pipetting or inverting. Aliquot 2 mL of cell suspension into each well of the 6-well dish. Incubate the...anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution Volume of Lentivirus...cells in each well. When calculating titer, only consider wells with less than 40% fluorescent cells. Titering...

  50. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...gels. They are shared through Addgene without licensing restrictions. Use these instructions to prepare... genome, many applications in biotech, and is considered the most studied Gram-positive bacterium, many...University of Minnesota recently deposited a comprehensive set of plasmids that can be used for either ...
Showing: 451 - 500 of 881 results