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We narrowed to 313 results for: POR C

Showing: 41 - 60 of 313 results

  1. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    Published
    Oct. 12, 2017, 2:26 p.m.
    ...PCR or by evaluating visual phenotypes. Improving C. elegans fluorescent protein tagging with SapTrap ...called SapTrap to improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one...protein tagging systems For those who do not work with C. elegans, several groups have designed modular toolkits...by the Hornung Lab, allows users to easily create C-terminal tag fusions of endogenous genes in human ...plasmid containing the homology arms and an N- or C-terminal tag. The two PCR reactions are then mixed...line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag, YFP... mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled...

  2. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    Published
    May 13, 2025, 1:15 p.m.
    ...Gilmour, K. C., Adams, S., Thornhill, S. I., Parsley, K. L., Staal, F. J., Gale, R. E., Linch, D. C., Bayford...L., Quaye, M., Kinnon, C., Ancliff, P., Webb, D. K., Schmidt, M., von Kalle, C., Gaspar, H. B., Thrasher... PMCID: PMC7120651 Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander... Mansour, M. R., Schwarzwaelder, K., Bartholomae, C., Hubank, M., Kempski, H., Brugman, M. H., Pike-Overzet...PMC189872 Schröder, A. R., Shinn, P., Chen, H., Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration...sequences, which facilitate integration of this portion into the host genome. The packaging plasmid contains...more detailed description). Since the integrated portion does not contain any of the viral packaging genes...

  3. What's the Best Way to Elute and Store Your Plasmid DNA?

    Type
    Blog Post
    Published
    June 4, 2020, 1:22 p.m.
    ...common solution is to keep your plasmid at -20°C or even at -80°C, in this case your preparation can be eluted...periods of time, especially at room temperature or at 4°C. Storing DNA: temperature and longevity You should... stable for years. Plasmid DNA can be stable at 4°C or even room temperature for a short period, and there...preventing the activity of DNase. While this is very important to preserve the stability of your DNA, it will...

  4. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ...MMR activation.  The first way is to introduce a C/C mismatch at or within 6 base pairs of the edit site...-50% when activation of MMR is avoided Introduce C/C mismatches or silent changes in wobble codons to ...recombination genes at low temperatures (30-34˚C).  Shifting bacteria to 42˚C for 15 minutes quickly inactivates ...required for maintenance. A temperature shift to 42˚C not only allows for activation of the red genes needed...PubMed Central PMCID: PMC2790811. 5. Thomason, L. C., Sawitzke, J. A., Li, X., Costantino, N. and Court...system to modify your target DNA, you first electroporate a linear donor DNA substrate (either dsDNA or...Expression of lambda red recombination genes. Electroporation of substrate DNA and outgrowth of bacteria....

  5. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    Published
    July 25, 2017, 1:20 p.m.
    ...with C. elegans. Researchers commonly use rectal bacterial infection to induce tail swelling in C. elegans...also used a photoswitchable CDK5 in C. elegans. See figures 4B and C in Zhou et al for details on these...-ERK signaling in the hypodermis and gut cells of C. elegans causes the same phenotype. Activation of ... an ERK kinase translocation reporter with a mRuby2 fluorescent reporter (ERK KTR-mRuby2). The ERK KTR...control of kinase activity can provide greater spatiotemporal resolution than pharmacological or genetic ...everything downstream of MEK1), bypassing the upstream portion of the pathway and focusing on the downstream steps...protein of interest. If you plan to try this, it’s important to check that the function of the protein and ...

  6. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    Published
    Sept. 10, 2024, 1:15 p.m.
    ...Biddle, M. S., Alende, C., Fotouhi, M., Jones, C., Ayoubi, R., Southern, K., Laflamme, C., Virk, H., NeuroSGC...T. M., Brown, C., Bandrowski, A., Virk, H., Edwards, A. M., McPherson, P., & Laflamme, C. (2023). Scaling....154034.1 Laflamme, C., McKeever, P. M., Kumar, R., Schwartz, J., Kolahdouzan, M., Chen, C. X., You, Z., Benaliouad..., H. S., Biddle, M. S., Smallwood, D. T., Weston, C. A., Castells, E., Bowman, V. W., McCarthy, J., Amrani...Biddle, M. S., Wright, A. K. A., Sousa, J., Weston, C. A., Chachi, L., Roach, K. M., & Bradding, P. (2019... monoclonal antibodies. It is also important to seek out supportive data on candidate antibodies from ...t always indicate the presence of supportive data. To be supportive, the data must show that the antigen...

  7. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    Published
    April 6, 2017, 1:02 p.m.
    ...example gene targeted for tagging with GFP at the C terminus, resulting in one edited allele, or gene ... gene of interest and decide which terminus, N or C, to tag and which short amino acid linker to use. ...study the protein or structure of interest. For a C-terminal tag, as shown in the example above, we inserted...precisely where we need it, in this example, at the C-terminus of the gene of interest. To get the molecular...tagging mammalian cell lines, Drosophila cells, and C. elegans. In addition to generating fluorescent protein...lines can be found in the Cell Catalog on our data portal. The plasmids should be functional in many different...and the donor plasmid into the cells we use electroporation, a technique that briefly destabilizes the ...

  8. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    Published
    July 23, 2015, 2:30 p.m.
    ...recent models of C. elegans molecular biology are based on the standard worm genome. While C. elegans are ...list of active spaces near you.   Learning about C. elegans with the OpenWorm Project One such space ...project. The main goal of OpenWorm is to simulate C. elegans as a computer model. When I first heard about...deconstruct and understand this complexity using existing C. elegans mutants. For instance, we use a mutant known... funded by someone else (KittyBiome has been incorporated as a non-profit entity and so far most of their...

  9. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    Published
    March 21, 2019, 12:47 p.m.
    ... (AacCas12b) exhibits optimal DNA cleavage at 48 °C, a temperature incompatible to genome editing in human...Bacillus hisashii, BhCas12b that is functional at 37 °C but preferentially nicks the non-target DNA strand...nicking Cas9. This base editor can be used for A•T to G•C edits in protoplasts or plants of rice and wheat. ...containing these base editors Spatially and temporally regulated AAV-based genome editing In 2016, Jonathan...AAV-based genome editing system is regulated not only temporally with doxycycline, but it can also be regulated...

  10. 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...

  11. 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 ...

  12. 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...

  13. 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...

  14. 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 ...

  15. 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...

  16. 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...

  17. 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...

  18. 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...

  19. 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...

  20. 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 ...
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