...material to specific cells in an organism. Figure 1. Using viral plasmids to generate virus. A) Viral ...of certain genes that are required for viral replication. Thus, as a safety feature, viruses used in research...Instead, the genes that are required for viral replication are usually divided among many plasmids, such...

...Hyg Chen 86196 pYPQ141-ZmUbi-RZ-As Maize ubiquitin 1 none As Cpf1 Qi 91715 pKEE401 yes, cut S. pyogenes...Gene/Insert Selectable Marker PI Publication Base Edit Catalytically dead dCas9 fused to a cytidine deaminase...Insert Promoter Selectable Marker PI Publication Activate Catalytically dead dCas9 fused to a transcriptional...Insert Promoter Selectable Marker PI Publication Interfere Catalytically dead dCas9, or dCas9 fused to a transcriptional...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs ...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted...

...to determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images). Figure 2: AAV...typically prepared using endotoxin-free plasmid purification kits. Resources Browse lentiviral preps available...

... large inserts AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid 911E4 ... This construct is then transformed into AdEasier-1 cells , which are BJ5183 E. coli cells containing ...multiple viruses. The adenoviral backbone pAdEasy-1 is suitable for most purposes. For especially long...Glossary Packaging Vocabulary Term Definition pAdEasy-1 Adenovirus packaging plasmid that lacks E1 and E3 ... for viral transcription; Supplied by the pAdEasy-1 packaging plasmid or 911E4 packaging cell line Transfer...adenoviruses replication deficient? Typically yes, because the early genes necessary for replication have been...events creating replication-competent virus increases with each round of amplification. What is RCA? RCA...

...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...target site. Benefits of truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated...protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA...gRNA (20nt complementarity to target site) or truncated gRNA (≤15nt complementarity to target site). When...

...activity in single cells using translocation reporters. Nature protocols, 13 1, 155-169. PMID: 29266096. Find...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...which have previously been characterized. See Box 1 in Kudo et al. for more details on the design of the...phosphatase-accessible. Kinase Translocation Reporters (KTRs) Kinase Translocation Reporters (KTRs) are genetically...provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization...activity is enhanced, leading to cytoplasmic translocation of the reporter. As a result, the KTR is shuttled...nuclear export and import rates can influence KTR translocation. Work from the Covert lab suggests that these...

...Figure 1: A chart with MoClo kit recommendations for different modal organisms and applications.  ...Traditional Biology.” ACS Synthetic Biology, vol. 5, no. 1, American Chemical Society (ACS), 4 Nov. 2015, pp...., vol. 4, no. 9, American Chemical Society (ACS), 1 May 2015, pp. 975–86. Crossref, https://doi.org/10.1021...Yeast Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. ...Editing in Plants.” BMC Plant Biology, vol. 20, no. 1, Springer Science and Business Media LLC, 23 Apr. ...Arabidopsis Mutants.” The Plant Journal, vol. 106, no. 1, Wiley, 25 Mar. 2021, pp. 8–22. Crossref, https://...Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media LLC, 12 July ...

.... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples...    Figure 1: Factors to consider when creating a pooled library amplification protocol. Created...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...ask for an amplification protocol with your deposit. This is because repeated amplifications of pooled ... an amplification protocol developed for each individual pooled library; generalized amplification protocols...Plasmids Variable Region Size DNA per Amplification Amplification Type Link to Protocol CRISPR Knockout... this blog, we’ll discuss how to develop an amplification protocol for your pooled library deposit. When...

...in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids...the deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...DE3. For Phagocata morgani and Phagocata gracillis, the process is a little more complicated. These strains...Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis. There are 11 different ... coli. Thus, to get the dsRNA into Phagocata morgani and Phagocata gracillis, one has to purify the dsRNA...

...preps, while other applications require large amounts of high quality DNA. Figure 1 illustrates the three... quantity.   Figure 1: Plasmid preps fall into different categories based on their relative qualities...volume ranges of culture as their input (Table 1). When you feel like you need just a bit higher DNA yield...kit for example. These kits are designed to handle ~1–5 mL of liquid culture. Using a higher volume can ...beads and a suitable magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep size Amount...Amount of culture Approximate DNA yield** Miniprep 1–5 mL 5–50 µg Midiprep 25–100 mL 20–200 µg Maxiprep...column-based DNA purification methods perform well in terms of quality and yield. Bead-based purifications tend to...

...the determination of amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...run in triplicate.  The nice thing about qPCR is that since it measures relative quantification - that... not need to be run in triplicates. ddPCR is an extremely accurate application, even in areas of the genome...Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications Resources on Addgene.org...

...series to increase binding capacity. Figure 1: Purify DNA using glass filters. Image: Kim and Morrison...a gravity-based column plasmid maxiprep kit takes 1-1.5 hours to complete, while this syringe-based method... solution. NaI has two purposes in this protocol: 1) it solubilizes both DNA and agarose, and 2) it helps...property and developed a protocol that uses Whatman No. 1 paper or even paper towels to purify DNA and RNA from... DNA purification without a kit as well as one way to reuse silica columns from DNA purification kits....consider some ways to purify DNA without a kit. DNA purification kits have advantages: they are convenient and...because you’ve run out of columns. Kit-less DNA purification methods avoid many of the drawbacks of kits ...

...one of three different inherited genes: Presenilin 1, Presenilin 2, and APP gene.The majority (>90%) of...loci seem to be associated with the disease and implicate a wide range of cellular processes. The vast majority... consists mainly of managing symptoms through medication and surgery. PD primarily involves the malfunction...available from Addgene, sortable by type of genetic modification and target species. You can also find our guide...Sun et al. Nat Methods. 2020 Oct 21. Glutamate indicators with improved activation kinetics and localization...A multicolor atlas for whole-brain neuronal identification in C. elegans. Yemini et al. Cell. 2021 Jan...window) A news website and information resource dedicated to helping researchers accelerate discovery and...

...by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct ... alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...Neuron-specific expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability ...genetic underpinnings of these processes has been complicated by the lack of precise genetic tools for modulating...recent advancements and offer commentary on their application to neuroscience research. What is CRISPR-based...accessible to neuroscientists, not all neuroscience applications require such optimized tools. This is elegantly...

...Element On or off PI 26429 pLenti CMV rtTA3 Blast (w756-1) Lentiviral reverse tetracycline-controlled transactivator...Lentiviral, miRNA expression (PRIME) system for application in knockdown of gene expression at a single copy...

... writing for the Addgene blog, sharing Chemistry Cat memes was how I used social media as a scientist....needed to start using Twitter. Sharing Chemistry Cat memes use to be how I used social media as a scientist...how scientists connect on Twitter that I realized 1) a lot of scientists are using social media professionally... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...of interest. Hopefully through these links, I’ve: 1) convinced you social media can positively impact ...remotely as an Addgene Science Communication Intern. Developing science communication skills, while also learning...

...CMV-EGFP PDF 2.3 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system...computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes, ...off-target sites. This algorithm allows for the quantification of both non-homologous end joining (NHEJ) and...number of potential off-targets, target's genomic location, and genome annotation. Available genomes are ...sequence, which can be an accession number, genomic location, pasted nucleotide sequence, or a sequence text...

...plasma HIV-1, improving life expectancy and quality for HIV-1 patients. However, ART is not an HIV-1 cure. ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number...currently infected with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies ...HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome...take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs...such a therapy, CRISPR/Cas9 would need to 1. excise the HIV-1 genome from every infected cell and 2. prevent...

... based on the HIV-1 virus. For safety reasons, transfer plasmids are all replication incompetent and may...shRNA‐expressing lentiviral plasmids, such as pLKO.1, use a U6 or H1 promoter in order to drive RNA pol...protocol for using the popular cloning vector pLKO.1. Trono Lab Resources: Lentivectors Toolbox and Maps...Desfarges S, Ciuffi A. Viruses. 2010. Jan;2(1):111-30. PubMed . Altering the tropism of lentiviral vectors...Curr Gene Ther. 2005. 5(4): 387-398. PubMed . HIV-1 Genome Nuclear Import Is Mediated by a Central DNA...Interchangeable: usually encodes for VSV-G Safety Safe. Replication incompetent: Uses 3 separate plasmids encoding...encoding various HIV genes. Safer. Replication incompetent and always SIN: Uses 4 plasmids instead of 3 and...

...editing efficiency and fidelity. Nature Communications , 13 (1), 1425. PMID: 35301321 Edraki, A., Mir,... DNA interrogation by SpRY-Cas9. Nature Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott...CRISPR-Cas9 to increase its specificity. Nature Communications , 9 (1). PMID: 30082838 Maddalo, D., Manchado, ...with an engineered chimeric Cas9. Nature Communications , 14 (1), 6175. PMID: 37794046 Small precision ...integration by homology-dependent repair. Nature Communications . 9 (1), 1133. PMID: 29556040 Chen, P. J., Hussmann...transcriptional reprogramming in bacteria. Nature Communications . 9 (1), 2489. PMID: 29950558 Fujita, T., & Fujii.... Biochemical and Biophysical Research Communications . 439 (1), 132–136. PMID: 23942116 Gilbert, L. A...