... compounds with various antimicrobial activities (1). She has developed a fast endophyte screening system...us coordinate and promote the awards. References 1. Martinez-Klimova, Elena et al. “Endophytes as sources...antibiotic” Biochemical Pharmacology 2017 Jun 15;134:1-17. PubMed PMID: 27984002. 2. Bilan, Dmitry S. et ...scientists work on projects spanning from the identification of plant compounds and insecticidal proteins...bacteria. Learn more about the different CRISPR applications in plants.     Transgenic yeast as source of... yeast capable of desaturating fatty acids. Identification of Insecticidal Proteins Linh Bao Ton (M. Sc...Arturo Rivera Solís, Universidad Autónoma de Yucatán, Mexico Dr. Rodrigo Arturo Rivera Solís, Associate...

...This complexity index ranges from 0-1, with a value of “0” indicating those markers are completely distinct...distinct from one another, and a value of “1” indicating those markers are identical and completely indistinguishable...detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...spectra to use for compensation.     Figure 1: The emission spectra and filters for FITC and PE ... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...

... basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...the CRISPR system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs...state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light... on itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide...outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required...Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing ...343–347. https://doi.org/10.1038/s41586-022-04470-1 Cofsky, J. C., Soczek, K. M., Knott, G. J., Nogales...

...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... be collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore... (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional resources on the Addgene blog  CRISPR 101...markers for plant transformation fall into two main categories: antibiotic/herbicide resistance and fluorescent...culture), which is great for a wide variety of applications. But some techniques require you to select against...these mutant plants. Using RUBY to simplify the identification of these mutants will speed up my work and ...student studying plant development and cell-cell communication in the Nimchuk Lab in the Biology Department...

... emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...cases Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if your deposit falls into...Addgene?" or contact us at [email protected] or +1 (617)-ADDGENE (233-4363) with additional questions...strain. Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if you’d like to distribute...page so that any data you enter is not lost.   Step 1: Sequences, Maps, and Files Please provide any full...a 1.5 mL microfuge tube at a concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you...in PubMed and choose the appropriate publication. This publication will be listed at the top of the final...

...Figure 1: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide...eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ORIGINAL ...red) or LbCpf1 (blue). Mismatch positions are indicated below each graph. Figure adapted from Kleinstiver... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...

...labeling of delicate axonal and dendritic processes not previously visible with Brainbow-1 and -2. The...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...subsequent recombinations cannot occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition to improving...temporal control without the need for germline modification, and enables Brainbow to be used in a variety...

... vectors are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential...systems have been developed over the years based on HIV-1; however, the 1st-generation vectors are never used...which has been shown to repress expression of HIV-1 LTRs (more information is available here). 2nd vs...lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated knockdown... looking for some additional information or clarification on these vectors. Read on to find the answers... thus decreasing the probability of creating replication-competent lentivirus (RCL) in your target cells...type or tissues; this is useful in gene therapy applications where a distinct subset of the cellular population...

...events by HDR can be generated more efficiently by (1) improving editing accuracy by preventing re-editing...specifically incorporate homo- or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking ...targeted alleles when gene editing.   Figure 1: Techniques to improve editing efficiency.   But...provided by blocking mutations. Essentially, you:  Step 1 - Introduce the blocking mutation together with the...developing novel iPSC-based model systems.     References 1. Cong, Le, et al. "Multiplex genome engineering using...many cell types and often results in biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous...

...), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...setting out of the lab (coffee, lunch or beer) and in 1:1 conversations. This creates a level of familiarity... other posts in our Science Mentors series: Part 1: "What Makes a Good Mentor?" and 6 More FAQs About...problems, develop general technical proficiency, communicate your work, maintain ethical standards and interact...websites listing alumi and it is easy to use publication records, university websites, ResearchGate and...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih... sequencing are more definitive, but visual identification is a good place to start before taking on more...resources are limited or for bioart and teaching applications. Find a chromoprotein for your experiment! ...events: The plasmid carrying the desired genetic modification integrates into the genome (Figure 3). Here,...of Allelic-Exchange Vectors for the Scarless Modification of Proteobacterial Genomes. Applied and Environmental...

...produce as many as 700 copies per cell. Figure 1: A plasmid map showing the standard features of a ...the origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all...   Types of origins of replication There are lots of origins of replication out there so, for simplicity...gene for replication (reference). **F1 is a phage-derived ori that allows for the replication and packaging...control elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself...are generally different from the those used to replicate the host's chromosomal DNA, but they still rely...readily at lower temperatures and allows the replication machinery room to come in and get busy making...

...vector at 1 x 1013 vg/mL titer), while AAV-PHP.S is typically used at a dose between 3 x 1011 and 1 x 1012...chosen capsid before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery...capsid applications. For example, AAV-PHP.eB is typically administered at a total dose between 1 x 1011...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023...easy to produce in high titer, but higher doses (>1 x 1012 vg total in mice) may increase the chances ...decision tree for selecting a capsid, while Table 1 gives a brief overview of published descriptions of...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...

...Table 1.  Table 1: Antibody data repositories and search engines Website Targets Application Purpose...which allow you to search many different antibody catalogs at the same time.  If you're looking for an antibody... specificities, from target-specific ones to application-specific ones to anything goes. We've shared ...

...allowed for the identification of proteins associated with the IRF-1 locus. Figure 1: Schematic of...the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting ...References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated ...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...

... genome which are required for replication and transcription (Figure 1). All viral genes are removed and...interested in genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula...cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly enhance our...reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed PMID:15298166. 3. Hoenen, Thomas, et al...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...

...plasmids to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap)... teaching me about AAV production and purification! References 1. Aurnhammer, C., Haase, M., Muether, ...plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and... repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ...cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...

...Figure 1), an altered epegRNA delivered with an enhanced pU6cm promoter, viral replicon amplification of ...of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-NC and ePEmax3...conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage of the self-labeling..., Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...

... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert a ... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the...in line 4 of Figure 4. Since the loxP sites are located in intronic regions, this gene will still be expressed...

...10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic...biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment...highly-visible beads on the cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking...enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it...scFvs for other targets or could be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...

...Fun fact: A 1 cm block of polymer can generate many meters of fibers!     Fig. 1 - Overview ... and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587-023-01833-5 Resources...channelrhodopsin 2 (ChR2), a blue light sensitive cation channel, fused to mCherry to the brain tissue. ... several months after implantation of fibers, indicating the system was indeed stable for continuous monitoring...interfaces with the entire body, so the obvious next applications are to expand the tool to different brain-organ...

...the Monoclonal Antibody Research Institute dedicated to: 1) developing new ways of generating antibodies... factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies ...target proteins of interest for labeling, quantification, purification, chromatin immunoprecipitation and more...Freedom: The Expansion of Data Sharing in Research Publications ...

...integration into CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-12449...NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created ...certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires...diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01780-w Additional...This reduces the chance of integration at random locations that could cause deleterious effects. It is important...multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the other houses the...

...and Therapeutic Purposes. Annu Rev Virol. 2014 Nov;1(1):427-51. DOI: 10.1146/annurev-virology-031413-085355...personalized medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy... Med Biol. 2014; 801: 687–693.  doi:  10.1007/978-1-4614-3209-8_86. PubMed PMID: 24664759. PubMed Central... time advances in human genetics led to the identification of several disease-causing genes, including... But it is the hard work and persistence of a dedicated team of scientists led by Dr. Jean Bennett (UPenn...-1250015778 7. Yu W and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina...

...) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV ...productivity but increases transgene expression. Sci Rep, 7(1), 5432. https://doi.org/10.1038/s41598-017-04054-4... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...structures, ITRs are prone to replication errors and disruptive mutations and truncations that can make vector ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as...are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...

...DNA with a PCR purification kit or with a phenol-chloroform extraction.    Fig. 1: Schematic overview...reserve a small aliquot, split into two parts to (1) run a DNA gel to confirm fragment size and (2)  incubate... let’s do a quick review. At this point you have (1) crosslinked your proteins of interest to any DNA ...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give... protein interactions, including epigenetic modifications. But it's not an approach for the faint of heart...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...

...Time: Topic) 0:00-1:00 : Intro 1:01-3:48 : Kingdom Kwapata's background and education 3:49-4:35 : Kingdom...Born to a family in Malawi that pushed science education, Kingdom Kwapata grew up to be one of the best...

...overlap with the negative control population (Figure 1). Based on where the GFP- population ends, you can...total cells present in the gate.   Figure 1: Example of yes-no flow plot of the (negative) control...for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example of a histogram plot ...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...

...the viral TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following...states was generated (Joung et al., 2023). Fig. 1: Barcoded TF library was packaged in lentiviral particles...atlas of directed differentiation. Cell. 2023. 186 (1) 209-229. doi: 10.1016/j.cell.2022.11.026 Joung J....unique barcodes for each TF help streamline identification. While the Zhang lab controlled their multiplicity...single TF was delivered to each cell, future applications could deliver multiple TFs to each cell, allowing...processes. For help with library transformation and amplification, refer to the Zhang lab’s previously published...published protocol and its relevant modifications on the pooled library webpage. Disease models Good disease...

...give your research more visibility.    Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg...constructs include proteins like nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and...Humana Press, pp 331–338 https://doi.org/10.1007/978-1-59745-181-9_23 Additional resources on the Addgene...other proteins that require post-translational modification may not be easily expressed in non-mammalian...constructs contain protein coding genes, tags, truncations, and codon optimizations similar to those found...are intended for the high level expression and purification of SARS-CoV-2 proteins for downstream experiments...wild type and codon optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned...

...plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid...and catenanes by agarose gel electrophoresis. Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...the right, with the entire monomeric sequence duplicated in tandem, resulting in a plasmid that is twice...

...between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one lox2272 site...cause of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox sites...recombination and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310...Vectors Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...

...or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal barcodes...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the...served as positive control (cis-ctrl). v1, vector 1; v2, vector 2; BD, binding domain; SDS, splice donor...)genome editing and gene therapy. Nat Commun., 14(1), 6578. https://doi.org/10.1038/s41467-023-42386-0...mice. B) Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s...ssAAV5s. C) Quantifications showing high microglia transduction efficiency for the indicated ssAAV5s. Figure...3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration, producing a second...

...that (1) the sequence is unique compared to the rest of the genome and (2) the sequence is located just...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...Biol 19: . https://doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, ...editing is most efficient when target sites are located in close proximity to the region to be edited. ... that were able to cleave target DNA sequences located upstream of either NGA or NGC PAM sequences (Kleinstiver...variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM sequences...

...the whole story Figure 1: Schematic representations of the indicated unbiased genotyping options.... and reproducibility of a study’s findings. Table 1: Biased Genotyping Options Technique Sequence Information...types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs in live ...magazine.scienceforthepeople.org/.     References 1. Fu, Yanfang, et al. "High-frequency off-target mutagenesis...next-generation sequencing." BMC genomics 15.1 (2014): 1. PubMed PMID: 25409780. PubMed Central PMCID: PMC4246457...off-target sites across the genome and pinpoint the location of mismatches based on the sequences of the genome...those relating to DNA structure (e.g. epigenetic modification, bulges) that may also impact off-target edits...

... dual-color control of neurons. Nature Communications, 12(1), 1–20. https://doi.org/10.1038/s41467-021...to consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Lehtinen, K., Nokia, M. S., & Takala... 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_2 Boyden, E. S., Zhang, F., Bamberg, E...channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from the archaebacteria Chlamydomonas ...stimulation. GPCR signaling GPCR signaling is a complicated process involving many downstream signaling mechanisms...break your experiments!  First, closely read the publication, including the supplemental data. You’re most...

... is conjugated to a fluorophore (Figure 1).   Figure 1: Comparison between how direct immunofluorescence...important considerations are to make sure your secondary 1) will recognize the species of your primary antibody...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to ...knowing that where you see fluorescence indicates your target’s location. Some other terms you might see associated...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...

... skills in science communication will be a positive asset throughout your career? 1. Make scicomm practice... the Communicating Science Conference series for graduate students. I believe that communication is the...skill in science communication over time.  Like most skills, great science communication comes with practice...able to communicate with their colleagues and peers. Scientists also need to be able to communicate to expert...need to communicate with non-scientists, to establish broader impacts for their work in education and policy...with opportunities to practice communicating.  Some fantastic K12 education initiatives like BiteScis (https...careers in science communication The strength of story telling Science communication: 9 strategies to get...

... the samples stored in our lentivirus tubes (Fig. 1). However, once thawed, there was a substantial reduction...in the pH of samples stored in our AAV tubes (Fig. 1). It’s important to note that the change in pH only...using the thawed virus within 24 hours. References 1. Murphy BM, Swarts S, Mueller BM, van der Geer P, ...improve our shipment processes. After reading a publication describing how short term storage on dry ice ...medium, which is buffered and contains the pH indicator phenol red. We froze samples in a -80°C freezer...closed dry ice storage container for 48 hours (to replicate how our viruses are shipped), where they would...exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or rather, not ...

...include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction from ...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic...editing applications.  Figure 4: Overview of CasMINI for genome engineering applications. Image ...stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies includes...to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling various targets... and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...

...unleashes its cargo – its own genome.   Fig. 1: Viral particle infection into a host cell. Image ...Vector Resources at Addgene! References and Resources 1. Roe T, et al. “Integration of murine leukemia virus...nuclear import of human immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. ...enough to ensure the virus can replicate and spread. Instead, viruses replicate by hijacking the host’s system...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn...people in nature, extra safety measures, like replication incompetent viral particles, are required. We...

...available from their original sources. Figure 1: Biological repositories that distribute mouse strains... from Reproducibility for Everyone. Case study 1: Cell lines Many of the reagents used to investigate...COVID-19: an observational study. The Lancet Microbe 1:e14–e23 . https://doi.org/10.1016/s2666-5247(20)30004...tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for ... Another example of reagents that have broad applications but have proven useful to the study of SARS-...vectors VSV-G and psPAX2, which were used in a publication by Xiuyuan Ou, et al. that characterized the ... requested these plasmids from Addgene. This publication then resulted in the development of a new plasmid...

...s pLKO.1 protocol. Pro-tip: this plasmid grows more slowly than standard plasmids. Find pLKO.1 - TRC cloning...experiments.  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David ...taken us on many adventures...From moving office locations three times, to opening our UK office in 2014,...cell types so it is useful in a wide range of applications. This plasmid was the top requested plasmid ...notice the “V2.0” at the end of this plasmid which indicates that there was a prior version of this plasmid...plasmid was called pLKO-Tet-On in the original publication but its name was subsequently changed to clarify...

...digital) signal at the end of the reaction (Figure 1). Partitions that did not contain any target DNA at...integrated through homology-directed repair (HDR) (1), or indel mutations resulting from non-homologous...Follow him on Twitter @AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base genome-edited...a gene of interest is one of the most common applications of precision genome editing, the remainder of...single molecule resolution of digital PCR allows classification of individual target molecules based on their...single wells of a 96-well plate without prior quantification. Comparatively, mismatch assays require between...

...standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants have been created, thereby...with finesse using tRNA suppression technology (Fig. 1) (Dumas, Lercher, Spicer, & Davis, 2015). By using...orthogonal labeling strategies are presented (Fig. 1) which can be carried out using Addgene plasmids. ...involved in skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored... transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method...cellular impediments. FRET has been used in many applications, including drug discovery by big pharma (i.e... acceptor doesn’t need to go to the same exact location as the donor. As long as some of the acceptor ...

... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you can...he still reads “#” as a “pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation...beforehand on which chromosome your docking site is located). This amounts to a small difference of two or ...throughout the lifespan of the fly to determine the implications of your genetic manipulations. You can produce...

...visualizing hM4D(Gi)-mCherry in fixed tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013...PMC3069789. 3. Kügler S, Kilic E, Bähr M. Human synapsin 1 gene promoter confers highly neuron-specific long-... preps. Cas9 is the headliner of most CRISPR applications and can be expressed from your cell line of ...Find Ready-to-Use Lentivirus Find Ready-to-Use AAV Catch up on Chemogenetics ...

...genetically targetable fluorescent indicators. Nature protocols. 2006 Aug;1(3):1057-65. Resources on Addgene.org...want a low affinity calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements... to a red fluorescent state only by simultaneous (1) user-controlled illumination with violet light and... a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...the sensors discussed below fall into this GECI category.   For calcium sensors, one size doesn...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...

...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced...facilitating recovery of edited animals. Figure 1: In the background are 504 restriction digests of ...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x... 3xFLAG epitopes into other, non-linked loci; pha-1(e2123) mutant worms are perfectly viable at 15 ºC,... repair pathway. Reagents required to perform pha-1 co-conversion are available through Addgene. In 2019...Mello lab also reported that modifying the 5’ modification of oligos used to generate linear dsDNA repair...Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. ...