...DNA, the CTD is also sometimes called the PAM-interacting (PI) domain. The guide RNA is bound primarily...most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, so it associates... less stringent recognition mechanism that can tolerate mismatches in this region. The crystal structure.... As mentioned above, however, Cas9 sometimes tolerates mismatches in the next 8-10 bases after perfect...this region of Cas9 also allows the protein to tolerate RNA:DNA mismatches between base pairs 18 and 20...Each new structural insight also spurs the next generation of engineered CRISPR enzymes with their own tradeoffs...Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization...

...incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes ...could detect knock-in of tags at a target site. Overall, 64% of researchers were unhappy with the new method...the water. On the bright side, successful indel generation and epitope knockin was achieved by a few respondents...ssDNA leading to inefficient downstream editing. Overall, the real take-home message thus far, based on ...form a complex with a 5’-P-ssDNA guide at room temperature. Until that time, those who have been lucky with...Pooran Dewari is a postdoc at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. His research interests...

...that Mobile-CRISPRi is transferable into multiple bacterial species including several involved in human disease...and is stable and active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components...highly conserved glutamine-fructose-6-phosphate aminotransferase (glmS) gene in the recipient bacteria due ...Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease (S. aureus... library strains was measured after 6 and 12 generations with or without induction of CRISPRi knockdown...

... (cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...consuming to prepare the 40% PEG solution. PEG takes several hours on a heated stir plate to dissolve and requires...carefully layered and then the viral suspension generated during AAV production is overlaid. Following ultracentrifugation... comparing to an AAV standard curve, which is generated from an ITR-containing plasmid. This whole process... always be included. While the physical titer generated by qPCR is useful, I test a few different concentrations...inverted terminal repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2....

...experiments require some consideration of FP properties. It is important to look out for general problems associated... judgement by simply “looking at them”. Modern cameras and image analysis software are highly sensitive...microscopy cannot be interpreted as direct molecular interaction without further investigation. One fluorescent...microscopy method commonly employed for analyzing interactions between two molecules more closely is Förster...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...

... copy number variation (CNV) assays. There are several varieties of these, but in this blog post, we're...length of the amplicon. As the probes can have several different markers, it can also be used to run multiplex...Do you need to call exact copy numbers or just general trends? How confident do you need to be in your...Addgene blog   Droplet Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications...Applications Resources on Addgene.org Protocol: Polymerase Chain Reaction (PCR) Protocol: How to Design a primer...SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination ...

...the conference After using your elevator pitch several times at the conference, you might notice some ...larger side? At large conferences, chances are that several talks will be going on at once in different rooms...a conference, be sure to practice beforehand. Generally, two types of people will visit your poster: those...have “tweep ups” where you can meet those you’ve interacted with on science Twitter in real life. If there...the tweep up (and for the entire conference, in general). After the conference Review your favorite conference... considered networking, but here are some more general tips. Before the conference Networking is key even...

... Their focus at the lab is on plant-pathogen interactions, and her aim is to produce constructs and edit...biosensors designed to elucidate the molecular interactions that go on between plants and their pathogens... share and educate and enable plant science in general. We are funded by a charity and since we are making...elements anymore. The kit contains sequences for several different fluorescent proteins. Visualizing fluorescence...piece of DNA. We do a lot of protein-protein interaction work and we’ve gotten some tools back from Addgene...Twitter account recently inspired by an academic in literature who gave a talk to the women’s research network...

...infection; however this would not be great for a gene therapy vector! For viral vectors, only the protein for...viral vector particles are made while missing the polymerase gene may seem like a mystery. But it’s simple...transcription and translation. Here, we will discuss several aspects of the viral “life cycle” in order to understand...step 3), but note also that it is worthwhile to generalize the idea of “transduction” to mean any transfer...et al., 1973). Interestingly, retroviruses are generally only “pseudodiploid” in that — while they have... M., & Bishop, J. M. (1973). RNA-directed DNA polymerase of Rous sarcoma virus: Initiation of synthesis...

...incorporated a discovery-based lab on planarian regeneration into one of my own courses, I was excited to...phosphorylation on the enzyme’s structure, function, and interaction with other proteins.   It is important to...be authentic – students should be attempting to generate new knowledge that will be of genuine interest...and bench work to identify regions of MDH that interact with citrate synthase and presented their findings...increasing diversity and inclusion in STEM disciplines (Bangera & Brownell, 2014; Duboue et al., 2022), as they...classroom.   References and Resources References Bangera, G., Brownell, S. 2014. Course-based undergraduate...

... Major restaurant chains including Chipotle and Panera Bread have pledged to eliminate the use of antibiotics...number of phagemids would be needed for an in vivo therapy. Another strategy for CRISPR antimicrobials combines...replication-competent phages and antibiotics. Phage therapy fell out of fashion with the introduction of antibiotics...Rodolphe Barrangou. “A CRISPR design for next-generation antimicrobials.” Genome Biol. 15(11):516. (2014...Ido, Miriam Manor, Ruth Kiro, and Udi Qimron. “Temperate and lytic bacteriophages programmed to sensitize... phage therapy...

... both vector amplification with low fidelity polymerases and the cost of synthesizing longer oligos, which...using the same three step protocol:  Fragment generation - Any linear DNA fragments can be assembled in... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...simple cloning procedures. Use high fidelity DNA polymerase This will minimize random errors during PCR: ...aliquots of PCR master-mix, containing buffer, polymerase and dNTPs, can be stored frozen: just defrost...mutagenesis by placing homologous ends on DNA using polymerase chain reaction." Biotechniques 10.1 (1991): 62...

...reporter enzyme. For multicolor staining, careful consideration must be used when selecting which antibodies...meticulously screening, optimizing, and characterizing several anti-mouse and -rabbit nanobodies. If you’re interested...described in Pleiner et al, the key steps for generating nanobodies are outlined below; no alpaca required...due to their N-terminal His14-bdNEDD8 tag. After several washes, this tag can be cleaved by the bdNEDP1 ...nanobodies from the column. bdNEDP1 protease can be generated using plasmid pDG02583. Label the Nanobodies: ...Maleimide-conjugated HRP can be found here and several maleimide-conjugated dyes are also commercially...

...genotypic and phenotypic artefacts. The life science literature is a litany of false interpretation in cell culture...Down-Regulation of HIF-1α by NDRG2 Expression Attenuates Proliferation and Invasion in Renal Cancer Cells, Marker-independent...out by the commercial repository. Repositories generally ensure cell line identity and purity making it...to the original as possible and so any results generated are reproducible and reliable. When you order ...which can be stored in liquid nitrogen long term, preferably some of these vials will be stored in a separate...identity. The Nature Publishing Group recently reiterated its strict stance on the matter. Funding bodies...

...was first developed in 2001, with successive generations developed ever since. GCaMP6 was offered through... added jGCaMP7 and eventually jGCaMP8, later generations of GCaMP6, to the service. While they were both...jGCaMP8 per year from 2016–2023. All three generations are regularly requested and a valuable part of...size of the incremental improvements between generations: jGCaMP8 allows for the “picture” of a train ... former approach and involves reading primary literature, soliciting information from requestors and depositors...Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

...ricin toxicity genes. The screen also discovered several genes previously not associated with ricin regulation...known ricin regulation genes compared to results generated using non-targeting guides as controls. While ...sites. Results from the screen also reproduced several previously observed characteristics that influence...activity: 1) mismatches closer to the PAM are less tolerated than mismatches more distal to the PAM, and 2)...off-target activity, Morgens et al drew some generalizable conclusions about off-target activity, demonstrate...Michael C., et al. "A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility...

...circuits and dynamics. FPs are generally bright, stable and well tolerated by cells. They can be used for...neither disturb POI folding nor its ability to interact with protein partners within the cell. However...can affect protein folding and protein-protein interactions. Also, overexpression of FPs can trigger protein...ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with facile ...monster” fluorescent proteins (smFPs). smFPs are literally composed of FPs fused to multiple epitope tags...

... to write, to read efficiently, to search the literature, and to synthesize a large volume of information...appreciate the discussion sections of the primary literature as a resource that both summarized results and... sure if I should cite reviews or the primary literature or both. I ended up citing both because I used...the scope and the content. Submit your article! Overall, writing a review can be overwhelming and challenging...Thompson, and Thomas D. Gilmore. "Histone acetyltransferases and histone deacetylases in B-and T-cell ...Addgene Blog Writing Scientific Manuscripts: Literature Searching, Reading, & Organizing Tips for Improving...

...soon find yourself encountering flow data in the literature. Data reported from flow cytometry experiments...work is done in flow cytometers, and output is generated through measurement of events. An event is something... machine is plotted as a dot on an X,Y axis to generate a dot plot (Figure 1). Clustered events are called...                         Figure 1: While the plot generated often looks like the graph on the left, it might...intersection of X,Y. For flow plots, however, that is generally not true. Because of the variations in fluorescent...Of course, this is quite easy to do on a plot generated in Biorender! While a helpful visualization tool...

...deliver all the protein and RNA components, and several sgRNAs must be screened to identify effective ones...Schematic of the Cre-dependent SPH construct for generating the SPH mouse. (B) AAV plasmids used to express...gene expression. Gene isoforms refer to mRNAs generated from the same gene but with different untranslated...regulation — at least, more straightforward than generating cDNA for each gene isoform individually. To highlight...their genomic DNA sequenced. Guide RNAs targeting several known and some unknown regulators of neuronal differentiation...Although cDNA overexpression approaches may still be preferable for individual gene activation in select in vitro...

...candidate regulatory kinase is known, it’s possible to generate non-phosphorylated recombinant protein and perform...Creating phosphoserine-incorporated proteins The generation of pSer-incorporated proteins requires a molecular...position. Practical tips for your experiments To generate as much pSer-incorporated protein of interest ...be optimized for parameters such as induction temperature and length of induction, which will vary between...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine... effects of pSer on enzyme function, protein interaction, etc. by comparison to non-phosphorylated or ...

...that only 11% of the academic research in the literature was reproducible by their groups. Finally results...copes with the growing amounts of digital data generated in labs. While many labs approach the situation...are available on the market today, ranging from general note taking tools, to more scientific field-based...From pictures to excel sheets, researchers today generate vast amounts of digital data during their work...within your lab? In every lab, there are certain hierarchies and restrictions to data access. If these are...walk you through your ELN’s features and this interaction will give you an idea of how responsive and supportive...

...member at Albert Einstein College of Medicine. Several sets of near-infrared fluorescent proteins (NIR...the NIR light coming from your source and to your camera.  Two-photon image of miRFP670nano3-expressing...reported widely used dimeric iRFPs (Table 1). In general, the monomeric state of a probe leads to less interference...al., 2018). For detection of protein-protein interactions, we recommend using NIR split reporters based...detect spatiotemporal dynamics of protein-protein interactions. Nat Methods 11, 641-644 (2014). Filonov, G....reporter for in vivo imaging of protein-protein interactions. Cell Chem Biol 20, 1078-1086 (2013). Shcherbakova...

...suggests there are hundreds. This only includes general lab strains designed for subcloning or protein ...ATPase that is essential for recombination and general DNA repair Reduces plasmid recombination, increases...Δleu mtl1 DH10B Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine...)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ- DH5alpha   General cloning and storage of common plasmids, blue/white...galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and plasmid maintenance, blue/white screening...(TetR) TOP10 Streptomycin MC1061 derivative. General cloning and storage, blue/white screening. F- mcrA...

...produce molecular biological tools like Taq polymerase, T7 polymerase, EcoRI, and various fluorescent proteins... elucidating the molecular mechanisms behind interactions between crops like potatoes and their microbial...that enable E. coli and Citrobacter freundii to generate sugars from cellulosic biomass. Dr. Duedu would...construction of new plasmids. Dr. Duedu will also use polymerases produced using plasmids from Addgene to test ...

...unpredictable loud noises and flashing lights - that sent average, control mice into depression. In case you were... skeletal muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into...between the muscle and brain, which may have therapeutic implications, say Hyo Youl Moon and Henriette...psychiatric and cognitive conditions as well as neurodegenerative diseases.” I don’t know about you, but I think...Pettersson AT, Ferreira DM, Krook A, Barres R, Zierath JR, Erhardt S, Lindskog M, Ruas JL. doi: 10.1016...

...for general audiences. So far, we are beginning to make an impact on society and science literacy. We ... Addictive Brain and science communication for general audiences. How does The Addictive Brain address...science, especially disease related topics. Neurodegenerative and brain disorders are frequently overshadowed...“manipulate” could mean “illicit tampering” to general audiences while “manipulate” could mean “worked...

...become resistant to drugs, particularly targeted therapies,” said Kris Wood of Duke University Medical Center...that are frequently implicated in cancer cell proliferation, survival, differentiation, and apoptosis. For...Rather, they relied on published studies in the literature to curate the perfect library, obtained the appropriate...researchers, and cloned them into lentiviral vectors to generate a kit which is now available at Addgene. “We called... cancer therapy...

...Plasmids 101 will provide you with an overview of general molecular biology knowledge and techniques, and...Site (MCS) Short segment of DNA which contains several restriction sites allowing for the easy insertion...later work.     How do scientists use plasmids? Generally, scientists use plasmids to manipulate gene expression...Green Fluorescent Protein (GFP) Plasmids 101: Luciferase Monitor the level of a chemical in a particular... viruses that can be used in research or for therapeutics Addgene has compiled various educational resources...

...besides structure and isotype determination, don’t generally affect antibody applications like ELISAs or Western...the receptors are named after the isotypes they interact with, so IgG isotypes bind to Fcγ receptors; IgE..., ADCC can be induced through a very similar interaction between IgE antibodies and eosinophils, where... induce?  Fc receptors are intriguing from a therapeutic standpoint. Engineered monoclonal antibodies ...phagocytosis for effective macrophage-based cancer immunotherapy.Sci. Adv.8,eabl9171(2022).DOI:10.1126/sciadv.abl9171...