...genome engineering methods for C. elegans were released, signaling a new era in C. elegans research. Homology...of custom genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification...the University of North Carolina has made CRISPR in C. elegans even easier - now, one can generate a fluorescent...-based gene tagging approach Multiple features of C. elegans have made CRISPR/Cas9 so successful in this...previously used a transgene selection strategy for their C. elegans/CRISPR experiments, but this strategy required... make it possible fluorescently label proteins in C. elegans, they inserted the SEC into a synthetic intron...system is both simple to use and highly efficient for C. elegans genome modification. Figure 2: Schematic...

...recognized by reliable antibodies. It is derived from the C-terminal region of the human proto-oncogene c-Myc. ... on the Anti-c-Myc [9E10] page for more details. Image credit: Addgene.   Find anti-c-Myc antibody...potassium channelrhodopsins Myc-tag antibody anti-c-Myc [9E10] now available! Improved voltage indicator...and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies with (magenta) and without...10.1101/2021.09.17.460684.)   Myc-tag antibody anti-c-Myc [9E10] now available! by: Ashley Waldron The majority.... Since the generation of the first anti-c-Myc [9E10] (Even, et al., 1985), the Myc tag has become one...pulled up over 13,000 plasmids! Our recombinant anti-c-Myc [9E10] (isotype IgG2a) antibody is currently recommended...

... L L T C G D V E E N P G P P2A: (GSG) A T N F S L L K Q A G D V E E N P G P E2A: (GSG) Q C T N Y A...plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is...ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between...between the Glycine and Proline residues found on the C-terminus meaning the upstream cistron will have a ...the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are thought to function... plasmids listed in the table above (and their related plasmids) are designed to have one or more of the...fluorescent reporter for easy detection.5  Just released: Addgene's Plasmids 101 eBook And now for some...

...PMC3223083. 6. McInnes, J. L., Forster, A. C., Skingle, D. C., & Symons, R. H. “Preparation and uses of...PMC164612. 11. Liou, Y.-R., Wang, Y.-H., Lee, C.-Y., & Li, P.-C. “Buoyancy-Activated Cell Sorting Using Targeted...Uses of biotin in molecular biology Biotin is a relatively small, water soluble molecule that does not interfere...fusion in which AviTag is added to either the N or C terminus of the protein. BirA has been shown to effectively...of some other common applications and links to relevant papers and webpages are listed below: EMSA (Electrophoretic...

... and C-C’) that give the feature its characteristic T-shape. The arrangement of the B-B’ and C-C’ palindromic... Tian, W., Liu, C., Lian, Z., Dong, X., & Wu, X. (2017). Deletion of the B-B’ and C-C’ regions of inverted...contains three palindromic sequences (A-A’, B-B’, and C-C’ regions) and forms a T-shaped structure. The Rep... Conatser, L. M., Lue, V. M., Dobbins, A. L., Li, C., Hirsch, M. L., & Samulski, R. J. (2020). Adeno-Associated... D. V. (2023). AAV- based vector improvements unrelated to capsid protein modification. Front Med, 10,...

...3rd 4 78,637 Broad GPP Humagne Set C and Humagne Set D 172650 (Set C) 172651 (Set D) Knockout Human Doench... for genetic screening experiments, due to the relative ease of designing gRNAs and the ability to modify...gRNAs By Species Mammalian Bacteria Drosophila Plant C. elegans Yeast Zebrafish Xenopus CRISPR Resources ...pneumoniae M. tuberculosis M. smegmatis Green monkey ( C. sabaeus ) Kaposi's Sarcoma-associated Herpes Virus...3rd 4 2,852 Bonifacino Lab Human ubiquitination-related proteins CRISPR KO library 174592 Knockout Human...

...CCL1 chemokine (C-C motif) ligand 1 I-309, P500, SCYA1, SISe, TCA3 CCL11 chemokine (C-C motif) ligand 11...chemokine (C-C motif) ligand 13 CKb10, MCP-4, MGC17134, NCC-1, NCC1, SCYA13, SCYL1 CCL14 chemokine (C-C motif...chemokine (C-C motif) ligand 19 CKb11, ELC, MGC34433, MIP-3b, MIP3B, SCYA19 CCL2 chemokine (C-C motif) ligand...chemokine (C-C motif) ligand 20 CKb4, LARC, MIP-3a, MIP3A, SCYA20, ST38 CCL21 chemokine (C-C motif) ligand...CCL24 chemokine (C-C motif) ligand 24 Ckb-6, MPIF-2, MPIF2, SCYA24 CCL25 chemokine (C-C motif) ligand 25...chemokine (C-C motif) ligand 26 IMAC, MGC126714, MIP-4a, MIP-4alpha, SCYA26, TSC-1 CCL27 chemokine (C-C motif...CCL28 chemokine (C-C motif) ligand 28 CCK1, MEC, MGC71902, SCYA28 CCL3 chemokine (C-C motif) ligand 3 ...

...in fact, permeable to CO2 at low temperatures (-80°C) and once in the tube, the CO2 can alter the pH of...pH indicator phenol red. We froze samples in a -80°C freezer and then moved them to a closed dry ice storage...samples to controls that were only stored in a -80°C freezer and not exposed to CO2/dry ice. All samples...then tested whether returning the tubes to the -80°C freezer after storage on dry ice allowed the CO2 to... (Fig.4). To test this, we froze the tubes at -80°C and then moved them to closed storage on dry ice for...for 48 hours. We then moved the tubes back to a -80°C freezer for various recovery periods between 0 and... samples to controls that were only stored at -80°C. We then thawed the tubes after the indicated incubation...

...?" Many thanks to our guest, Darrell Kotton. Dr. Kotton is the David C. Seldin Professor in the Department...about Darrell, his research, and his focus on open science. Listen to the interview with Darrell Kotton...Introduction to Darrell Kotton and the Boston University Center for Regenerative Medicine. Darrell describes...low-risk but reliable projects. Chapter 9: Using stem cells as in-vitro models of disease. How Darrell is using...Darrell Kotton is the director of the Center for Regenerative Medicine at Boston University Medical School... School. Darrell’s research focuses on the lungs, and, among other projects, using stem cells to develop...develop in vitro models for lung disease. Darrell strives to promote open source biology in his own lab, ...

...editing toolkit to allow C to U edits using their RESCUE (RNA Editing for Specific C-to-U Exchange) system...RESCUE-S) offered ~76% on-target C-to-U editing while reducing off-target C-to-U edits by ~45% and off-target...lab published a paper on an RNA editor that allows C to U edits (RESCUE). We’ll walk through how these ...adjusted the structure of the guide RNA by introducing a C opposite the target A to be edited. This change specifies...residues that contact the RNA substrate resulted in 15% C-to-U editing activity. They then performed sixteen...cytidine deaminase activity. While RESCUE can perform C-to-U edits, the authors note that it retains its adenine... that RESCUE is most active in a 30 nt guide when C or U base-flips are present across from the target...

...structures based on morphology. Autophagosomes are relatively easy to identify: double-membraned structures containing...comprised of two tandem fluorescent proteins: a relatively pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive...connected by a 9 amino acid linker that’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of...are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott, M., ...traditional method for studying autophagy. This method relies on the identification of autophagic structures ...lysosome, since both Atg8p and LC3 are degraded or released once an autophagosome fuses with the vacuole or...

...activity on NAG PAMS and reduced activity on NGG PAMs relative to wild type. A new genome-wide CRISPR knockout...) Strategy for leak-free transcriptional control. C) Iterative transduction workflow overview. Image from...nucleases identified, functions at temperatures up to 43 °C and can be used for genome editing in moderate thermophiles...opportunities for investigating genetic mechanisms related to morphogenesis, cell fate specification, and ...the internal control (Fig 1). Because this method relies on the fluorescence ratio between the 2 fluorophores...

... amplification in the host cell. This enables relatively high and long-term expression of the reprogramming...the reprogramming factors- OCT3/4, SOX2, KLF4, and C-MYC [1]. For the derivation of clinical grade hiPSCs...vectors that encoded six factors - OCT3/4, SOX2, KLF4, C-MYC, LIN28 and SV40 large T antigen (SV40LT). The ...of TP53 enhances iPSC generation as does replacing C-MYC with L-MYC [10, 11]. While the mechanisms underlying...understood, the addition of an shRNA for P53 and replacing C-MYC with L-MYC in the episomal reprogramming cocktail...patients are reprogrammed less efficiently, no such correlation has been found between the age of the patients...Models for Disease & Open Science: Interview with Darrell Kotton Four Factors that Differentiate the Stem...

...thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes. Ligation Dilute... Let's assume that your favorite vector has a relatively limited MCS (BamHI - EcoRI - SalI) and you want...off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy the EcoRI...oligos in a 1.5mL microfuge tube. Place tube in 90-95°C hot block and leave for 3-5 minutes. Remove the hot...

...fragments: N-terminal BirA* (N-BirA*) and C-terminal BiraA* (C-BirA*). These two fragments are fused to... are enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated proteins. One ...also which fragment of BirA*, either the N- or the C-terminus, the bait proteins were tagged with. It’s...These interactions are often modeled as binary relationships, i.e. protein A interacts with protein B; but...transient PPIs. Many PPI identification techniques rely on protein interactors to stay in close contact ...

... with 40–60% G/C content are most likely to be successful, although sequences with G/C content outside...of the 3′ extension of the pegRNA should not be C. A C base is speculated to base pair with G81 of the..., J., Oyler-Castrillo, P., Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A.,...Figure 3: pegRNAs whose 3′ extension begins with a C base can cause unwanted base pairing (left), while... Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). ... Knipping, F., Ravisankar, P., Chen, P.-F., Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, ..., Anzalone, A. V., An, M., Newby, G. A., Chen, J. C., Hsu, A., & Liu, D. R. (2022). Engineered pegRNAs...

...2018). For C>T and A>G base editors, the intended edit must be in a 5 - 10 nt window relative to the PAM...target sites that code for amino acids close to the C’ terminus of the protein, to maximize the chances ...bystander edits are possible if there is another target C or A in the window. Another technology, prime editing...doi.org/10.1038/s41587-020-0561-9 Dempster JM, Pacini C, Pantel S, Behan FM, Green T, Krill-Burger J, Beaver...JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang P-Y, Daley G, Church G (...introduction of a specific mutation, one generally relies on homology directed repair (HDR) to incorporate...along with the single cell clone (although this is rarely done). When targeting a dsDNA break for HDR, the...

...Time: ~40 minutes Equipment required: 37°C heat block and 62°C heat block Approach: Viral RNA is extracted...range Time: ~1 hour Equipment required: 37°C water bath and 42°C water bath Approach: RNA is extracted from...10.1038/s41596-019-0210-2 Lucia, C., Federico, P.-B., & Alejandra, G. C. (2020). An ultrasensitive, rapid...Valois, E., Solley, S. C., Braig, F., Lach, R. S., Baxter, N. J., Kosik, K. S., Arias, C., Acosta-Alvear, D...incubation  Time: ~90 minutes Equipment needed: 37°C heat block, LED blue light illuminator or UV light... in a range of temperatures varying between 10-50°C. Visit Addgene's COVID-19 Plasmids and Resources ...Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan C-Y, Guevara H, Wadford DA, Chen JS, Chiu CY (2020) ...

...While this method can be time-consuming, it is relatively effective in reducing off-target effects since...still unexpectedly affect nearby genes (Figure 1A-C). Find CRISPR plasmids at Addgene! Knock-in: Introducing...site (TSS) deletion. (B) Mutations of splice sites. (C) Removal of an exon or a large genomic fragment. (... doi:10.1080/15476286.2014.996067 Basak J, Nithin C. Targeting Non-Coding RNAs in Plants with the CRISPR-Cas...

... 25°C. At low temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies...genomes Compared to humans and mice, flies have a relatively simple genome. They have four chromosomes: the...females do not. Also, males have sex combs on their forelegs which are black, bristly hairs. Females do not...