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We narrowed to 321 results for: POR C

Showing: 51 - 100 of 321 results

  1. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    Published
    Sept. 28, 2023, 1:15 p.m.
    ...Yamada, K., Kuznetsov, I. A., Rand, E. A., Ochs, C. C., Jaber, Z., Gardner, K. H., & Chow, B. Y. (2022...eLife.84364.  Meiring, J. C. M., Grigoriev, I., Nijenhuis, W., Kapitein, L. C., & Akhmanova, A. (2022)...., N., Ononiwu, F., Pal, D., Manikas, J., Taveras, C., Hall, N. A., Silva, J. D., Freshour, J., & Hehnly...j.ceb.2015.08.005.  Tei, R., Bagde, S. R., Fromme, J. C., & Baskin, J. M. (2023). Activity-based directed ...structure, separate chromosomes during mitosis, support transport of organelles and vesicles, and more. Although...microtubule-cutting sword Microtubules are one of the most important components of the cytoskeleton. They provide cells...demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given the long list of...

  2. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...Only about 10 amino acids can be deleted from the C-terminus and about 5 from the N-terminus, but this... standard practice to attach the GFP to the N- or C-terminus of another protein. This may, however, interfere...consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence (-CaaX). Both modifications...when GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated the alternative...the reasons that this works well is that the N- and C-terminus of GFP itself are relatively close, thereby...G-alpha subunit fusions that do not tolerate N- and C-terminal fusions (Adjobo-Hermans, 2011). Structural...that generates the chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and ...

  3. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    Published
    Oct. 22, 2015, 2:30 p.m.
    ...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent... the UAS/reporter gene, the same bait and prey libraries can be screened using both N- and C-terminal ...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the ...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system (...sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing...

  4. A Look Back at One Year of Plasmid Sharing for COVID-19 Research

    Type
    Blog Post
    Published
    July 12, 2021, 1:15 p.m.
    ...doi.org/10.3390/v12050513 Hsieh C-L, Goldsmith JA, Schaub JM, DiVenere AM, Kuo H-C, Javanmardi K, Le KC, Wrapp...plasmids used for this study at Addgene which include a C-terminal deletion in the spike protein to improve ...Zengel JR, Sathyan V, Nguyenla X, Van Dis E, Catamura C, Yamashiro LH,  Begeman A, Stark JC, Shon DJ, Fox ...Wrapp D, Lee AG, Liu Y, Chou C-W, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Park J, Wang...around the world, the spike protein has had many opportunities to evolve. Several variants are now circulating...different spike protein variants that can be incorporated into pseudoviruses. The lab used these pseudoviruses...Addgene to better understand which human genes are important for SARS-CoV-2 entry into human lung epithelial...

  5. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    .... W., Déjean, V., Dodson, C. A., Wallace, M. I., Mackenzie, S. R., Timmel, C. R., & Hore, P. J. (2016)...Polali, S., Anderson, E. D., Bell, A. M., Tzouanas, C. N., Avants, B. W., & Robinson, J. T. (2019). Magnetic...doi.org/10.1021/jacs.3c01076.  Wheeler, M. A., Smith, C. J., Ottolini, M., Barker, B. S., Purohit, A. M., ...Beenhakker, M. P., Kucenas, S., Patel, M. K., Deppmann, C. D., & Güler, A. D. (2016). Genetically targeted magnetic.... Andrew and Maria were encouraged by previous reports that described magnetic responses of cryptochrome...reproduced their EGFP findings since the initial report in 2023.)     Figure 1: Fluorescence intensity...frequent collaborator and is happy to give advice and support for follow-up studies. “We don’t have a vision ...

  6. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    Published
    Aug. 27, 2020, 1:15 p.m.
    ...information, you can fuse each FP fragment to the N- and C-terminal end of each PIP and empirically test which...R, Björklund M, Ahr D, Ahmed AW, Spinelli L, Brun C, Basler K, Merabet S (2018) Generation of a versatile.... eLife 7. https://doi.org/10.7554/elife.38853 Hu C-D, Chinenov Y, Kerppola TK (2002) Visualization of...doi.org/10.1101/pdb.prot076497 Kim Y, Jung JP, Pack C-G, Huh W-K (2018) Global analysis of protein homomerization...https://doi.org/10.1101/gr.231860.117 Kodama Y, Hu C-D (2012) Bimolecular fluorescence complementation ...conditions when compared to FRET. However, it’s important to note that BiFC is irreversible — FP fragments...interact or not? — FRET is preferable when the temporal dynamics of PPIs are the focus of study. Many...

  7. Antibodies 101: Producing Recombinant Antibodies

    Type
    Blog Post
    Published
    Aug. 13, 2024, 1:15 p.m.
    ...PMC3725456. Schwarz H, Zhang Y, Zhan C, Malm M, Field R, Turner R, Sellick C, Varley P, Rockberg J, Chotteau...aliquot of our recombinant antibodies is stored at 37 °C for a period of two weeks. After this time, quality...33151027; PMCID: PMC7665837. L'Abbé D, Bisson L, Gervais C, Grazzini E, Durocher Y. Transient Gene Expression...Sederstrom D, Steinmann D, Mozziconacci O, Schöneich C, Freund E, Ricci MS, Carpenter JF, Lengsfeld CS. Do...for further processing. It may be helpful to incorporate regular feeds during this incubation time to ...immunohistochemistry, and immunocytochemistry. It’s important to note that some antibodies are better suited...Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of...

  8. Hot Plasmids: Spring 2025

    Type
    Blog Post
    Published
    June 3, 2025, 1:15 p.m.
    ...expressing iGluSnFR3, iGluSnFR4f, and iGluSnFR4s. C) Spike-triggered averages for single action potential-evoked...tagging: Choose untagged or add a versatile N- or C-terminal HA tag. Selectable and stable: A puromycin...integrase-based approaches. Find STITCHR plasmids here! Fell, C. W., Villiger, L., Lim, J., Hiraizumi, M., … Abudayyeh... Cenik, B. K., Aoi, Y., Iwanaszko, M., Howard, B. C., Morgan, M. A., Andersen, G. D., Bartom, E. T., &.../doi.org/10.1016/j.molcel.2024.11.007. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina...and PV-positive cells out of all BiPVe3-dTom cells. C) Confocal stack of a biocytin-filled cell expressing... receptors and transporters. The intensity-based Glutamate-Sensing Fluorescent Reporter (iGluSnFR) designed...

  9. Hot Plasmids - June 2019 - Optogenetics, Acoustic Reporter Genes, microRNAs, and the CRISPR-Cas9 system CHIME

    Type
    Blog Post
    Published
    June 6, 2019, 12:41 p.m.
    ...chain of BoNT/B into two fragments (BotNT-N and BotNT-C) and joined each fragment with one part of an established..., and showed that it can alter motor responses in C. elegans. Find the split BoTN/B plasmids at Addgene...collection of reporter plasmids is amenable to Golden Gate assembly for turning the single-input reporters into...Neuron. 2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive imaging of microbes in mammalian...contributed by Lukas Morgan  Listen to the acoustic reporter gene podcast segment In recent years, scientists...heavily on fluorescent protein genes. These optical reporters have limited deep-tissue performance, and sometimes...Mikhail Shapiro’s lab has developed four acoustic reporter gene (ARG) plasmids to better monitor gene expression...

  10. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ...cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen for ITR recombination with...flexibility since I can store the cell lysates at -80 °C for up to 6 months before purifying the AAV (Choi ...in one long day, or the virus can be stored at 4 °C and buffer exchanged the next day. Refer to the AAV...production and purification! References 1. Aurnhammer, C., Haase, M., Muether, N., Hausl, M.A., Rauschhuber...virus from the cell pellet alone. If yield is important, or if the viral particles of the AAV serotype...

  11. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, Gogolok S et al. 2017. ...available for human and mouse genomes only and deals with C-terminus epitope tagging. If you work with other model...your crRNAs using other web-based design tools (for C-terminus tagging, pick a crRNA that cuts 8-15 bp downstream...site (within ~8-15 bp from the stop codon for C-terminus tagging) GC content of the crRNA (aim for 40-60%... genotyping can tell if HDR completely failed. If C-terminus tagging potentially disrupts protein function... O'Duibhir E, Quinn N, Tyrer A, Leitner MC, Plumb C, Kalantzaki M et al. 2018. An efficient and scalable...Desktop Genetics .For ssODN design, remember to incorporate PAM-blocking mutations (e.g. convert the NGG ...

  12. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ...alternating between 5 min at -20°C (or -80°C) followed by 5 min at 37°C (each repeated 5 times) works well...with any protein purification, but overnight at 30°C is a good start. Prepare a lysis solution containing... PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine M. Szymanski. "Exploring the...PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine M. Szymanski. "Development ...to probing with cells in order to optimize this portion of the assay before attempting it with a random...

  13. To Each HIS Own

    Type
    Blog Post
    Published
    March 2, 2023, 2:15 p.m.
    ...histidine amino acids on the N- or C- terminus of a protein. Neither the N or C terminus is preferable over ...histidine residues expressed in frame on the N- or C-terminus of a recombinant protein of interest. The...need to decide whether to add the tag to the N- or C-terminus and how many histidine residues to include...tended to be very large and often required the incorporation of a cleavage site and a post-purification cleavage...very pure sample. Because binding strength is proportional to the length of the tag, with longer tags you...

  14. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    ..., Kadmon, J., Raja, C., Chibukhchyan, A., Ramakrishnan, C., Inoue, M., Shane, J. C., McKnight, D. J., ...Mattis, J., Tye, K. M., Ferenczi, E. A., Ramakrishnan, C., O’Shea, D. J., Prakash, R., Gunaydin, L. A., Hyun...A., Pieper, F., Sims, R., Tenedini, F., Bergs, A. C. F., Bendifallah, I., Zhou, F., Zeitzschel, N., Ahlbeck...individual types of GPCRs with greater spatial and temporal control than is achieved by traditional pharmacologic...photoswitchable, orthogonal, remotely-tethered ligands (PORTLs). OptoXRs are chimeras of the extracellular domain...change shape with light exposure to activate them. PORTLs use a similar concept, except the ligand is attached...

  15. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ...rise of literary giants such as Isaac Asimov, Arthur C. Clarke, James Blish, and John W. Campbell. This period...combination of the four bases of DNA – A, T, G, and C. In this movie Ethan Hawke’s character revolts against...editors (CBE) use a cytidine deaminase to introduce a C→T change at precise locations in the genome. This ...JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR (2019) Search-and-replace...Correction: Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 559:E8...420–424 . https://doi.org/10.1038/nature17946 Lenay C (2000) Hugo De Vries: from the theory of intracellular...editing technology led to the development of many important medical and agricultural products. But it also...

  16. Hot Plasmids - October 2020

    Type
    Blog Post
    Published
    Oct. 13, 2020, 1:15 p.m.
    ... synaptic proteins Fluorescent protein system for C. elegans Fluorescent timers for studying cell cycle...native locus, but introducing large genetic changes in C. elegans with CRISPR can be difficult since it requires...can you use CRISPR to introduce large FP genes into C. elegans? By using a split FP approach. Split-wrmScarlet...together, they fuse and fluoresce. The team engineered C. elegans cell lines to express wrmScarlet1-10 unattached...grown in high concentrations of IL-3 show a high proportion of blue fluorescence, indicating cell proliferation...they could not proliferate. Eastman et al. Cell Reports 2020. https://doi.org/10.1016/j.celrep.2020.107804...

  17. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    Published
    Dec. 14, 2021, 2:15 p.m.
    ...02708-5 Hrit J, Goodrich L, Li C, et al. (2018) OGT binds a conserved C-terminal domain of TET1 to regulate...89–92. https://doi.org/10.1038/301089a0. Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking...https://doi.org/10.7554/eLife.34870.  Kaas GA, Zhong C, Eason DE, Ross DL, Vachhani RV, Ming G-L, King JR...sigh. The problem is that many of these studies report correlations without clearly testing whether a ...Sapozhnikov and Szyf 2021, Charlton et al. 2020). Most importantly, TET proteins affect transcription independent...

  18. Plasmids 101: Biotinylation

    Type
    Blog Post
    Published
    Nov. 15, 2018, 1:50 p.m.
    ...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...fusion in which AviTag is added to either the N or C terminus of the protein. BirA has been shown to effectively...of carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl...primary antibody respectively. Avidin bound to a reporter is then added to the reaction for easy detection... experiments. During detection, a biotinylated reporter enzyme that is pre-incubated with free avidin ...protein of interest leading to amplification of the reporter signal and easier detection. This strategy is ...

  19. Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation

    Type
    Blog Post
    Published
    May 21, 2020, 1:15 p.m.
    ...labeling of the cell population. Figure 2: Dense (c), intermediate (d), and sparse (e) labeling of the...References: del Valle Rodríguez A, Didiano D, Desplan C (2011) Power tools for gene expression and clonal ...Feng Q, Zhou Y, Zeng S, Ren M, Jiang S, Ni H, Zhou C, Gong H, Luo M (2018) Cell-type-specific and projection-specific...was developed by Thomas Clandinin’s lab, and incorporates several common genetic tools in Drosophila (...: a method to genetically manipulate precise proportions of cells. bioRxiv 788679; https://doi.org/10.1101...

  20. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ...fragment of CIB1 and it was fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of ...fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714) halves, rendering...split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription of the...used the bacterial phytochrome BphS which produces c-di-GMP in response to far-red light (730 nm). This...VP64 which in turn transcribes N-Cas9. The N- and C-Cas9 fragments then dimerize to form a functional ... from A. sativa (Bubeck et al., 2018). LOV2 has a C-terminal helix that unfolds when stimulated by blue...Bietz A, Waldhauer MC, Börner K, Fakhiri J, Schmelas C, Dietz L, Grimm D, Correia BE, Eils R, Niopek D (2018...

  21. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ... 79628 (C) Cv (capsid) N and C 79633 (N) and 79641 (C) NS1 (var: W98G) N and C 79635 NS3 C 79637...79637 NS2B C 79636 NS4A N 79640 NS4B C 79639 NS5 C 79632 (N) and 79631 (C) PrM N and C   Detecting... for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions. Kim, et al. Mol Biol Cell. 2016...streamline CRISPR/Cas9-based genetic tag insertion in the C. elegans genome, Erik Jorgensen’s lab from the University...paramagnetic ferritin protein were tethered to the C-terminus of TRPV4 (a pressure-sensitive channel). ...Rodríguez-Laureano L, Albanese SK, Gradia S, Jeans C, Seeliger MA, Levinson NM, Chodera JD. bioRxiv preprint...et al. Cell. 2015. PubMed PMID: 26627737 pORTMAGE: A portable method for bacterial genome engineering ...

  22. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    Published
    Sept. 9, 2014, 8:20 p.m.
    ... 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...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 ...along with a reporter, such as a fluorescence gene or a resistance gene. These reporters allow you to ...sometimes advantageous, particularly when only a portion of the plasmid is packaged for viral delivery, ...expression levels between two or more genes is important. How do multicistronic vectors work? Scientists...single transcript. The two strategies most widely incorporated into plasmids for research purposes are described...

  23. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ...target sites that code for amino acids close to the C’ terminus of the protein, to maximize the chances ... absence of dsDNA breaks (Rees et al., 2018). For C>T and A>G base editors, the intended edit must be ...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 (...sequence are important considerations for designing your gRNAs. For indels, it's not so important what location...nom. Me love gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment ...

  24. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    Published
    Oct. 18, 2018, 12:37 p.m.
    ... added to the PCR mix to remove parental DNA (37 °C for 15 mins). This enzyme specifically cuts methylated...region, with a melting temperature (Tm) of around 60 °C for all primers. The length of the homologous region...recombination. We normally use a Tm between 47-55 °C for the homologous region (calculated using Oligo ...product. For example, by combining multiple primers, a C-terminal epitope tag can be moved to the N-terminus...pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested so far...use of this pathway as a cloning tool was first reported over 25 years ago (Douglas and Howard, 1991, Bubeck...cloning strain has failed so far. What is most important is the transformation efficiency of the strain...

  25. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    Published
    Nov. 12, 2020, 2:15 p.m.
    ...cells. For example, Henry Lester’s lab optimized the C. elegans GluCl ion channel genes for expression in...Santana-Quintero LV, Katneni U, Simonyan V, Kimchi-Sarfaty C (2017) A new and updated resource for codon usage ...–34 . https://doi.org/10.1038/nsmb1031 Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and ...doi.org/10.1093/nar/28.1.292 Sauna ZE, Kimchi-Sarfaty C (2011) Understanding the contribution of synonymous... in a particular organism. Why is codon usage important? Translation and protein synthesis relies on transfer...deliver amino acids to the ribosome where they are incorporated into the growing polypeptide chain. One part...even been linked to some diseases. It has been reported that 5-10% of human genes contain a region where...

  26. Using AAV for Neuronal Tracing

    Type
    Blog Post
    Published
    Aug. 9, 2018, 1:04 p.m.
    ...J.G., Wohnoutka, P., Gerfen, C.R., Koch, C., Bernard, A., Dang, C., Jones, A.R., and Zeng, H. (2014). A ..., J. M., Vandenberghe, L. H., Haute, C. V., Thiry, I., Deroose, C. M., Mortelmans, L., Wilson, J.M., Debyser... Ravi, N., Wu, W.-L., Sánchez-Guardado, L., Lois, C., Mazmanian, S.K., Deverman, B.E., and Gradinaru, ...Winslow, B., Cain, N., Mihalas, S., Wang, Q., Lau, C., Kuan, L., Henry, A.M., Mortrud, M.T., Ouellette,... E., Tanaka, M., Kearney, M., Chattree, G., Yang, C. F., Shah, N.M., and Mooney, R. (2017). Identification...Res 406(1-2), 317-321. PubMed PMID: 2436717. Towne, C., Schneider, B.L., Kieran, D., Redmond, D.E.Jr., and... facilitate transport: retrograde transport relies on dynein, while anterograde transport relies on kinesin...

  27. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    Published
    Feb. 4, 2025, 2:15 p.m.
    ...References and Resources References Braun, C. J., Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design...Sousa, A. A., Tsai, S. Q., Bengtsson, N. E., Lööv, C., Ingelsson, M., Chamberlain, J. S., Corey, D. P.,...M. J., Joung, J. K., Breakefield, X. O., Maguire, C. A., & György, B. (2019). High levels of AAV vector...CRISPR screens rely on lentiviral systems, a fact supported by the wide array of lentiviral-based CRISPR libraries...locations that could cause deleterious effects. It is important to note, however, that while AAV rarely integrates..., CRISPR may increase this rate. Specifically, portions of the AAV genome can integrate at double-stranded...

  28. Plasmids 101: How to Name Your Plasmid in 3 Easy Steps

    Type
    Blog Post
    Published
    Feb. 13, 2014, 1 p.m.
    ...resistance, b) the promoter that drives the insert, and c) any other selection markers (for use in other cell...pBACKBONE-Flag-hGene If there was also an EGFP fused to the C-terminal of your insert you would list it after the...identify what your plasmid contains and other important information. Tip: A lowercase "p" is often used...simple piece of information can often convey many important details. Once you know the backbone a plasmid ...

  29. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ... Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019). ..., X., Chen, W., Martin, B. K., Calderon, D., Lee, C., Choi, J., Chardon, F. M., McDiarmid, T. A., Daza...Kissling, L., Marquart, K. F., Schmidheini, L., Solari, C., Balázs, Z., Krauthammer, M., & Schwank, G. (2023...-024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, X., van der Weide, R. H., Morris, B., van... In our study, we used the TRIP (Thousands of Reporters Integrated in Parallel; Akhtar et al., 2013; Schep...Chromatin Position Effects Assayed by Thousands of Reporters Integrated in Parallel. Cell, 154(4), 914–927....

  30. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    Published
    Dec. 7, 2020, 2:53 p.m.
    ....1126/science.abb2920 Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH (2020) CRISPR...and require only two transposase proteins (TnsB and C). While requiring fewer components for RNA-guided ...Rubin BE, Diamond S, Cress BF, Crits-Christoph A,  He C , Xu M, Zhou Z, Smock DC, Tang K, Owens TK, Krishnappa...genomes engineering In the Sternberg group’s initial report of VchINT, three separate plasmids and multiple...simple to use, users should keep aware of a few important details. For transposons to be specifically recognized...

  31. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...80 °C. Titer All titering is performed on lentiviral preparations that have been stored at -80 °C and ...Preparations are then aliquoted and stored at -80 °C. Titer Titering is either performed by Addgene or ...preparations are then aliquoted, frozen, and stored at -80 °C. Concentrated lentiviral preparations (“-LVC” catalog...expression and/or function. These data are sometimes reported or posted on the material page for the corresponding...recipient’s initial thaw will be accounted for in our reported titers. Lentiviral vectors are titered using a...

  32. Antibodies 101: Epitope Tags

    Type
    Blog Post
    Published
    May 11, 2023, 1:15 p.m.
    ...Epitope tags are short peptides introduced at the N or C terminus of a protein that are bound by antibodies...enterokinase, which recognizes the 5 amino acids on the C terminal of the tag. Of the most common tags, FLAG...not at all. The introduction of a tag at the N or C terminus of a protein may disrupt the biological function...destabilization can be brought about by the tag so it’s important to validate tagged protein functionality (Arribere...

  33. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    Published
    May 23, 2023, 1:15 p.m.
    ... Boeckman, J. X., Manning, C. F., Nguyen, J. T., Bechtold, H., Dumitras, C., Gong, B., Nguyen, K., Van...-GABA-AR β1 (white) and anti-GABA-AR β3 (green). (C) Hippocampus labeled with anti-Kv2.1 (magenta), anti-AnkyrinG... Panels C1-C3 show magnified detail of the box in C. Image adapted from Andrews et al., 2019 under CC-... J., Kandov, E., Lowekamp, B., Speranza, E., Chu, C. J., Gola, A., Thakur, N., Shih, R., Yao, L., Yaniv...protocol is a good place to get started.  One important consideration when deciding whether you can do...specific considerations to keep in mind. Most importantly, make sure you are using primary antibodies from...

  34. Hot Plasmids - September 2019 - CRISPR, Auxin Inducible Degradation, MerMAID Channelrhodopsins, and Genome Looping

    Type
    Blog Post
    Published
    Sept. 3, 2019, 1:15 p.m.
    ...please sign up here.        New toolkit for N or C terminal tagging of proteins for degradation using...has created a new toolkit of plasmids for the N- or C- terminal tagging of proteins with mAID and other ...cytidine deaminase  Target-AID-NG, which mediated a C to T conversion in the target sites adjacent to an...Earlier this year, the Hegemann lab and collaborators report their discovery of a new family of seven ChRs derived...

  35. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...4355596 King, S. R., Duggal, N. K., Ndongmo, C. B., Pacut, C., & Telesnitsky, A. (2008). Pseudodiploid Genome...A., Hunter, E., & Kappes, J. C. (1997). Functional RT and IN incorporated into HIV-1 particles independently...34372494. Prussin, A. J., Garcia, E. B., & Marr, L. C. (2015). Total Virus and Bacteria Concentrations in...

  36. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    Published
    June 1, 2023, 1:15 p.m.
    ... Sellnow, R. C., Boye, S. L., Coberly, B., Bennett, A., Agbandje-McKenna, M., Sortwell, C. E., Hauswirth...Backer, M. W. A., Brans, M. A. D., Luijendijk, M. C., Garner, K. M., & Adan, R. A. H. (2010). Optimization...Dudek, A. M., Pillay, S., Puschnik, A. S., Nagamine, C. M., Cheng, F., Qiu, J., Carette, J. E., & Vandenberghe..., J. Y., Han, J. H., Lee, H., Han, Y. E., Rah, J. C., & Park, H. (2020). Labeling dual presynaptic inputs...injection site  (Kanaan et al., 2017), axonal transport  (Murlidharan et al., 2014), and postsynaptic ... system, remember that some serotypes can be transported retrogradely and/or anterogradely along the axon...et al., 2014; Nieuwenhuis et al., 2021). It is important to note that this expression pattern can be affected...

  37. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    Published
    April 8, 2025, 1:15 p.m.
    ...independent blots. B–C) Graphs showing the relative intensity of the β-actin (B) or total protein (C) on the membrane...quantification in panel B is not especially linear. In panel C, two different methods of quantifying total protein...linear. Note the different y-axes between panels B and C, indicating that β-actin is less sensitive as well...BioRender.com. Loading controls are even more important for semi-quantitative western blots. No pipetter...in the same lane, and the resulting fraction is reported. Figure 2: Normalization with loading controls...equally strong, so the normalized value simply reports which POI band is stronger. The right gel has exactly...abundance on your membrane, which would be directly reported by band intensity. Researchers use later steps...

  38. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    Published
    Jan. 10, 2023, 2:15 p.m.
    ... was reduced by growing seed stocks at 30°C compared to 42°C. Screen multiple colonies to isolate the ...19763938. Maucksch C, Bohla A, Hoffmann F, Schleef M, Aneja MK, Elfinger M, Hartl D, Rudolph C. Transgene expression...Growing the culture at a lower temperature such as 30°C or optimizing the incubation time can help maintain...long-read sequencing techniques, such as Oxford Nanopore sequencing or PacBio SMRT sequencing, (which differ...long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio SMRT sequencing. These longer... plasmid multimerization troubleshooting tips. Portions of this figure were made with BioRender. ...AE. Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch...

  39. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    Published
    Oct. 19, 2017, 1:54 p.m.
    ...optimum of 30 °C and is therefore inefficient in mammalian cells (usually grown at 37 °C). Smart molecular...identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance... receptor (ER) ligand binding domain (LDB) to the C-terminus of FLP or Cre (pCAG-CreERT2 #14797). These...FLP-FRT. In addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed...These differences in recombinase efficiency are important to keep in mind when deciding whether to use the... only occur at a specific time point? To allow temporal control, ligand regulated Cre and FLPe recombinase...control of Cre recombinase. After in-utero electroporation, this plasmid mix results in sparse labeling...

  40. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    Published
    Dec. 14, 2017, 2:08 p.m.
    ...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...into cells by electroporation. hiPSC colonies are obtained by 10 days after electroporation, most of which...over a decade back when Yamanaka and colleagues reported that it is possible to reprogram differentiated...generation of human iPSCs (hiPSCs) now provides an opportunity to develop and use patient-specific somatic cells...

  41. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

    Type
    Blog Post
    Published
    July 21, 2017, 12:48 p.m.
    ...21356876. 5. J. Chow, Z. Marka, I. Bartos, S. Marka, J. C. Kagan, Environmental Stress Causes Lethal Neuro-Trauma...PubMed PMID: 28704652. 6. V. Rodríguez, D. Didiano, C. Desplan, Power tools for gene expression and clonal...Neurosci. 9, 703–709 (2006). PubMed PMID: 16582903. 8. C. J. Potter, B. Tasic, E. V Russler, L. Liang, L. Luo...you to systematically study gene function with temporal control and cell-type specificity! The Gal4/...

  42. Antibodies 101: Flow Cytometry Controls

    Type
    Blog Post
    Published
    June 6, 2024, 1:50 p.m.
    ...starting point for compensation (Fig. 1B-C). However, it’s important to note that because you’re using beads... single color control for the APC channel. B) and C) A flow plot showing an APC single color control prior...prior to compensation (B) and after compensation (C) with APC-Cy7.   FMOs FMO stands for fluorescence...

  43. 7 Tips to Secure a STEAM Internship This Summer

    Type
    Blog Post
    Published
    Feb. 2, 2021, 2:15 p.m.
    ...and Rose C. St Pierre, founders of STEAMid. Internships open doors to a web of opportunities that the ...guest bloggers Roodolph (Roo) P. St Pierre and Rose C. St Pierre! Roodolph (Roo) P. St Pierre is currently...populations. Roo is the co-founder of STEAMid.       Rose C. St Pierre is currently a graduate student at Northeastern... not deter any student from applying to the opportunities of their dream.  To help students focus on the...below:  1. Eligibility check Scrutinize every opportunity prior to starting an application to assess your...unless you are applying for a Federal Government opportunity, which tends to be more detailed. Beyond the ...draft a personal statement, which is the perfect opportunity to paint a comprehensive picture of your academic...

  44. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    Published
    Feb. 24, 2015, 7:50 p.m.
    ...to fuse the activators instead of at the protein’s C terminus. With that new design in place, Konermann...Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki...Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann ...nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop...knockout screening). As the Zhang lab's Nature paper reports, “Here we have shown that the SAM system is robust...size of the backbone, transformations require electroporation. References & Further Reading: For more ...

  45. Deep Dive: qPCR

    Type
    Blog Post
    Published
    Sept. 29, 2022, 1:15 p.m.
    ...Annealing temperature should be 60-65°C For qPCR, the important primer variable is the annealing temperature... the primers.  Melting temperature should be ~60°C (between 50 and 65, with no more than 3 degrees between...curve will show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak...dimers. (b) A shoulder peak indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits...02217-xTaylor, SC; Nadeau, K; Abbasi, M; Lachance, C; Nguyen, M; Fenrich, J. The Ultimate qPCR Experiment...Zuo J, Dong X, Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin...step. Scorpion chemistry in particular has been reported to work well in the Lightcycler while being relatively...

  46. CRISPR 101: Non-Homologous End Joining

    Type
    Blog Post
    Published
    April 16, 2015, 3:45 p.m.
    ...Oh S, Tomkinson AE, Hendrickson EA, Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations.../10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, Scott DA... Yan M-Y, Li S-S, Ding X-Y, Guo X-P, Jin Q, Sun Y-C (2020) A CRISPR-Assisted Nonhomologous End-Joining...process involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways...chance of causing a frameshift mutation. Of some importance, the deletion can be less heterogeneous when ...

  47. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    Published
    April 16, 2024, 1:15 p.m.
    ...mDia1 and CP appearance at the filament tip from (a). c) Intensity traces at the tip of the filament from ...10.1021/cb900062k  Gautier, A., Juillerat, A., Heinis, C., Corrêa, I. R., Kindermann, M., Beaufils, F., & Johnsson...https://doi.org/10.1126/science.281.5374.269  Jing, C., & Cornish, V. W. (2011). Chemical Tags for Labeling... M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana, R. F., Urh, M.,...doi.org/10.1038/nmeth749  Strebinger, D., Frangieh, C. J., Friedrich, M. J., Faure, G., Macrae, R. K., &...amino acids to directly incorporate a fluorophore in your protein or to incorporate an alkene to be labeled... self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from Roger Tsien’s lab ...

  48. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ...at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, or a truly... Additionally, protein lysis should be done at 4 °C or on ice, whichever is more practical. If your lysing... truly lengthy incubation (30–60 minutes) at 37 °C, if you suspect aggregation in your prep. If your proteins...protein-based blockers degrade fairly quickly, even at 4 °C, so when in doubt, make up a fresh batch.  Antibodies...and time. Though the most common conditions are 4 °C overnight or room temperature for 1–2 hours, there...sample loaded Incubate primary antibody at 4 °C overnight Incomplete washing Ensuring wash is...under-transferring.  Membrane pore size Membranes come in different pore sizes for different proteins:...

  49. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    Published
    March 23, 2021, 1:15 p.m.
    ...interactions New auxin inducible degron system for C. elegans New CRISPR plasmids New items from the viral...tool-kit for the auxin inducible degron (AID) system in C. elegans By Alyssa Cecchetelli The auxin inducible... degron (AID) system has been extensively used in C. elegans since 2015 to conditionally degrade proteins...length can and should be varied as it can have important implications for the fusion protein effectivity...upon these original tools that allows for both spatiotemporal protein degradation and quantification of this...et al., 2021.   The AID system relies on a transport inhibitor response 1 (TIR1) expressed under a ...a desired promoter with a specific spatiotemporal expression pattern and an auxin inducible degron (AID...

  50. "Hall of Fame" AAV Enhancers from the Allen Institute for Brain Science

    Type
    Blog Post
    Published
    Sept. 9, 2025, 1:15 p.m.
    ...https://doi.org/10.1016/j.cell.2025.05.002 Hunker, A. C., Wirthlin, M. E., Gill, G., Johansen, N. J., Hooper...Larsen, R., Gore, B. B., Szelenyi, E. R., Trader, C., . . . Tasic, B. (2021). Enhancer viruses for combinatorial...Somasundaram, S., Miller, J. A., Kalmbach, B. E., Radaelli, C., Gore, B. B., Weed, N., Omstead, V., Bishaw, Y., ...10.1007/s00018-021-03903-w  Thomas, H. F., & Buecker, C. (2023). What is an enhancer? BioEssays, 45(10). https...evaluate the resulting expression of the fluorescent reporter, the brain tissue was extracted, sectioned, and...more neuroscience-focused tools in their Brain Map portal, and cell science-focused tools at the Allen Institute...expression from mouse to primate neocortex. Cell Reports, 34(13), 108754. https://doi.org/10.1016/j.celrep...
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