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...holds true in genome-wide screens.   Figure 1: Summary of the advantages of the new approach to ... in both screens, with the growth screen having a 1% false discovery rate and identifying >88% of previously...in Morgens et al. contains three types of guides: 1) targeting guides (blue), which target all protein-coding...off-target sites was profiled. Guides with exact or 1-bp mismatch off-targets had greater toxicity than ...characteristics that influence gRNA off-target activity: 1) mismatches closer to the PAM are less tolerated than.... When comparing the toxicity of guides that have 1-bp mismatch off-target sites, short guides had reduced...Knockout Library are available from Addgene! References 1. Morgens, David W., et al. "Genome-scale measurement...

... and investigation. References: 1. The problem of pseudoreplication in neuroscientific studies: is it...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...control and is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...two types of replicate controls: technical and biological. In general, technical replicates can be thought...

...the result was a simpler 1 protein - 1 RNA system.  Dr. Charpentier advocated for the crucial role of ...minutes, which was fun to watch. A simpler 1 protein - 1 RNA system Drs. Dounda and Charpentier met at...adaptive bacterial immunity and its potential applications in rewriting the genome. Their award acceptance... same Polish sub-dialect making their Skype communications extra fun) who shared first authorship on their... area will accelerate the launch of clinical applications. During the panel discussion, Dr. Mello took...for billions of years (…) Bacteria are really sophisticated”.     New to CRISPR/Cas9 Technology? Learn ...

...materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created ...EBNA1 Cells. Methods Mol Biol. 2018;1850:1-16. doi: 10.1007/978-1-4939-8730-6_1. PMID: 30242676. Frenzel...Neuroscience Research. Curr Protoc Neurosci. 2020 Dec;94(1):e106. doi: 10.1002/cpns.106. PMID: 33151027; PMCID...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...addgene.org Addgene's Antibody Purification Protocol Addgene's Video Antibody Purification Protocol Addgene's Antibody...cells can perform human-like post-translational modifications that bacterial and most yeast cells cannot. ...production system may depend on what downstream applications you are planning, cost, and the amount of protein...

...across scientific disciplines. Scientific Data, 8(1), 1–11. https://doi.org/10.1038/s41597-021-00981-0 Wright...occurrence during vector purification and formulation. Molecular Therapy, 12(1), 171–178. https://doi.org...excellent at infecting our target neurons (Figure 1). However, this one set of experiments didn’t provide...yet none of that data was published.    Fig. 1: Testing of three different AAV serotypes (AAV1, 8...Stimulation. Frontiers in Human Neuroscience, 14(December), 1–12. https://doi.org/10.3389/fnhum.2020.578687 Additional...caudal NST. (A-C) Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained... right AAV for your system or to successfully replicate published works. Of particular importance to these...

...range of titers. We usually dilute our samples 1:6 million to 1:25 million.  After making the dilutions, they...note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies (GC) per reaction. Since...In the image to the right, there is approximately 1 positive copy per microliter in the no template control...elongation time to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation...diagnosis." Emerging microbes & infections 7.1 (2018): 1-9. PubMed PMID: 29691363. PubMed Central PMCID: PMC5915492...take to achieve a clean NTC: Have a dedicated bench with a dedicated set of pipettes for ddPCR set-up. .... Each droplet contains the ingredients for amplification of the target DNA. This partitioning reduces...

..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...containing a phage origin of replication that can be packaged into replication-incompetent phage particles...essential processes: cell wall synthesis, DNA replication, or protein synthesis. Because they target such...

...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with the exception...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will...to additional protocols and plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "...

...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing a ...assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...weight of the proteins you’re interested in. Table 1 lists the most common types of gels for SDS-PAGE running...gel chemistry and/or specialty gel options. Table 1: Types of gels  Gel Type Protein sizes Running... Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...

...Science Communication Conversation in Science Communication The Purpose of Science Communication #1 Mistake...think about communicating science. Listen to the interview with Science in the News, Part 1 Podcast breakdown...supporters of science communication. To help you think about ways to effectively communicate your science, we...News at Harvard University. Effective science communication is required to promote public support for research... organization that works to promote science communication. Science in the News hosts a podcast, seminars...That’s right, #ActualLivingScientists, eager to communicate their science. In this first half of our two-...Mistake Scientists Make when Communicating with Non-Scientists How to Approach Different Audiences Listen...

...protocols. A typical ExM protocol (Fig. 1) follows this structure: Step 1: fix (and maybe label) your sample...microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1  Mikhaylova M, Cloin BMC, ...just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify ...16:71–74. https://doi.org/10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023...with an abundance of protocols and examples of applications from across disciplines now available. In this...experiment or evaluating ExM images in a new publication.         Why Expansion Microscopy?  Traditional...microscopes sounds great, right? But there is always a catch - some limitation of the technique that users should...

...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR sites...regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. ...expression of the human KRAS gene in mammalian cells. STEP 1: Generate an Entry Clone There are a few different...could use a vector like pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible pLIX_403. The chosen...the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction... Find Gateway cloning plasmids here! References 1. Chee JY, Chin CF (2015) Gateway Cloning Technology...attP sites of the donor vector. This reaction is catalyzed by the BP Clonase enzyme mix and generates the...

...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for...Handling Visualization Signal Amplification Application Overview Quantification Methods Capture Methods Visualization...Antibody Applications Antibody-based applications can be generally classed into four categories: Quantification... Read on to learn more about the applications common to each category, including an outline of the basic...these applications, visit the Antibody section of the Addgene Protocols page. Antibody Applications - Quantification...concentrates in the sample. Controls for quantification methods Quantification methods should be run with: a positive...

...Virus Replication. Essential Human Virology, 49. https://doi.org/10.1016/B978-0-12-800947-5.00004-1 Taylor... an envelope are called enveloped viruses (Figure 1). Like bacteria, only a tiny fraction of viruses are...are pathogenic (Balloux & van Dorp, 2017). Figure 1: A non-enveloped virus (left) and an enveloped virus...create far safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...

... gp41-1 Split inteins A small, fast split intein SpyTag003/SpyCatcher003 SpyTag/SpyCatcher Rapid covalent...the best performance in the most contexts (Figure 1). Together the features with optimal biological performance...described as Click Biology (Howarth, 2025). Figure 1: Modular assembly tools, alongside criteria for Click...tools can be found in Addgene's repository (Table 1). Figure 2: Adding function to My Favorite ...coupling. Created with BioRender.com.   Table 1: A handful of Click Biology tools available at Addgene...peptide (SpyTag) and protein (SpyCatcher) SnoopTag/SnoopCatcher SpyTag/SpyCatcher Rapid covalent bond formation...SnoopTag) and protein (SnoopCatcher); no cross-reaction with SpyTag/SpyCatcher, allows for iterative expansion...

...including yourself as the researcher.   Figure 1: Separation of retroviral packaging components onto distinct...these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar to AAV in that...lab coats are either recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus...required whenever there’s a splash or spray risk in BSL-1. In BSL-2 labs, eye protection is required any time...isopropyl alcohol, 0.5% peracetic acid, 10% bleach, or 1% iodine are useful. Some organizations or cities may...host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection... tools, thought was given on how to keep the replication of virus and its infectivity in check. Engineering...

... Podcast breakdown: (Time : Topic) 0:00-1:00 : Introduction 1:01-9:04 : What got Professor Cepko Interested... producer for the Addgene Podcast.     References 1.Tang, Jonathan C.Y., et al. “A nanobody-based system... PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “...PubMed PMID: 26258682. Find plasmids from this publication at Addgene. Resources on the Addgene Blog Read...

...comprised of 2 components: 1) an SB transposase, the enzyme required for catalysis of transposition; and 2...also important for social and regulatory reasons. 1.) The transposon consists of a mirrored set of inverted...likely to grow in the coming decades.  References 1. Kowarz E, et al. Optimized Sleeping Beauty transposons...T cells to target CD19. Immunol Rev. 2014 Jan;257(1):181-90. doi: 10.1111/imr.12137. PubMed. Find the ... Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells... though not all, of these defenses. For some applications of genome engineering - such as certain forms...to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion site.   The...

...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...assemblies are possible, the two sets of data together indicate that 35-pieces is a reasonable upper limit for...

... 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,...Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.” Genetics. 202(4)...