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We narrowed to 395 results for: POR C

Showing: 331 - 360 of 395 results

  1. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...either the PAM (changing one of the G’s to an A,T or C – but avoid NAG) or guide RNA target (“seed”) sequence...it is incorporated. We were surprised to see how rapidly the efficiency of mutation incorporation falls...contains the intended sequence changes to be incorporated into the edited genome. In mammalian cells such...these two strategies allows you to specifically incorporate homo- or heterozygous mutations. 1. Increasing...therefore strongly encourage any researcher to incorporate CRISPR/Cas-blocking mutations at both targeted... our frustration) in many cases we only saw incorporation of the blocking mutation. Despite the fact that... away from the cut site were not efficiently incorporated during HDR. We decided to systematically characterize...

  2. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    ...PubMed PMID: 7915669. Dominguez, Roberto, and Kenneth C. Holmes. "Actin structure and function." (2011). PubMed...genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important for...protein Groucho, a transcriptional co-repressor important in development. Groucho family proteins are found...Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest...video game character but rather to an extremely important gene in animal development. To understand where... Tinman, 2016). Bagpipe on the other hand, is important in the formation of the visceral mesoderm which...

  3. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    ...habitats - even 50 meters from Old Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs...number of systems that can be studied by providing support "for development of tools, approaches, and infrastructure...great for studying parenting strategies and make important ecological indicators since species live in water...Aiptasia - algae Breviolum minutum symbionts are important models for solutions to the coral reef crisis.... interesting biologically. They have economic importance as harmful weeds. The parasitic fusion is such...

  4. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...2.50021-0  Toh, W. K., Teo, Y. L., Tor, X. Y., Loh, P. C., & Wong, H. L. (2023). Development of constitutive...methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous...Paracoccus denitrificans, Pasteurella multocida, Porphyromonas gingivalis, Proteus mirabilis, Providencia spp...Xanthomonas campestris   In addition to incorporating broad host range replicons, recombinant vectors...can be expressed. For example, scientists can incorporate interchangeable antibiotic resistance cassettes...

  5. Genetic Code Expansion

    Type
    Collection
    ...synthetase subunit E. coli p-azido-L-phenylalanine (Azf) C. elegans ATG David Tirrell 62599 pKPY197 phenylalanyl-tRNA...(CePheRS) C.elegans p-azido-L-phenylalanine (Azf) C. elegans ATG David Tirrell 63177 pMarsL274G methionyl-tRNA... the codon), the amino acid from the tRNA is incorporated into the growing peptide chain. To expand the...aminoacyl-tRNA-synthetase (aaRS) that has been evolved to incorporate non-canonical amino acids (ncAAs). A plasmid ...the cells, the non-canonical amino acid can be incorporated using the existing protein translation machinery...translation machinery are needed in order to incorporate a non-canonical amino acid into the protein of... the place where the new amino acid would be incorporated. Other options, such a 4-base pair codons, have...

  6. Antibodies 101: Reading a Flow Plot

    Type
    Blog Post
    ...Barlow-Anacker, A., Bochkov, Y., Gern, J., & Seroogy, C. (2017). Neonatal immune response to rhinovirus A16...encountering flow data in the literature. Data reported from flow cytometry experiments can be a little...correlates with the occurrence of post-transplant opportunistic infection in liver and kidney recipients. World...

  7. CRISPR References and Information

    Type
    Collection
    ...activation). Genome support for bacteria ( E. coli, B. subtilis ), yeast ( S. cerevisiae ), worm ( C. elegans )...gRNA and HDR donor design. The current version supports SpCas9, SpCas9-VQR, and enAsCas12a Cas enzymes...Lab . Read more about it on the Addgene blog . CRISPOR (Link opens in a new window) This tool helps design...(2016) (Link opens in a new window) . Also try CRISPOR batch (Link opens in a new window) for lentiviral...input sequence and checks for off-target binding. Supports a range of Cas enzymes (Cas9 and Cas12) and organisms... or a sequence text file you upload. Currently supports a wide selection of organisms. From the Database...sequence and checks for off-target binding. Currently supports a wide range of organims. Developed by the Michael...

  9. Quick Guide to Near-Infrared Fluorescent Proteins

    Type
    Blog Post
    ... internal tags due to the proximity of the N- and C-termini. Latest miRFPnanos9 do not yield to (m)iRFPs...molecular brightness (Table 1).  NIR reporters and biosensors NIR reporters can tell us about cellular events..., D. & Michnick, S.W. An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions...FPs) were derived from natural photoreceptors incorporating biliverdin IXα (BV) autocatalytically. Fortunately...should be supplied (Baloban et al., 2017).  It is important to consider effective brightness of NIR FPs in... a cell and depends on the efficiency of BV incorporation by an apoprotein and protein expression level...higher apparent cellular brightness than previously reported widely used dimeric iRFPs (Table 1). In general...

  10. Evolution of Lab Techniques

    Type
    Blog Post
    ...1975): 503-517. PubMed PMID: 1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new prenatal test...although they are reported to have high error rates (10). It is projected that nanopore-based sequencing... similar experimental questions (see below). Importantly, although their role has changed, restriction...lengths because extension is terminated upon the incorporation of a ddNTP. Researchers perform four different...activity as fluorescently labeled nucleotides are incorporated, a method called pyrosequencing. The drive for...sequencing world, one up and coming innovation is nanopore-based DNA sequencing which reads the nucleotides...nucleotides of a DNA strand as it slips through a tiny pore by measuring differences in ion flow caused by the...

  11. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ... easy for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest...found in your gene of interest) and prevent incorporation of extensive sequences used for cloning into... your research to a variety of experimental opportunities. The plasmids in this kit have been used to ...library by in vivo functional screen." Scientific reports 5 (2015). BIOFAB plasmid set 3000 Series Confronted...are just the tool you need. The plasmids in this portion of the BIOFAB Plasmid Set contain terminators of...strengths centered within GFP/RFP bicistronic reporters characterized in Cambray et al., 2013. These terminators...Helena, et al. "Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation...

  12. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ...a variety of imaging conditions (155 μs and <0.1 °C in optimal conditions). While B-gTEMP itself is a ...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...plasmids here!   Farahani PE, et al. pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling...10.1101/2022.08.13.503850.  iGECInano NIR calcium reporter Near-infrared (NIR) fluorescent proteins promise...photochemical characteristics than previous NIR calcium reporters iGECI and NIR-GECO2G, and its small size (~1500...sensors Whatever your analyte of interest, it’s important to choose a biosensor that has a binding affinity...

  13. Plan Your Experiment

    Type
    Collection
    ...) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite of being ...promoter is typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive...vector or separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify... to target cells through microinjection or electroporation Transient expression of CRISPR components Expression...may alter protein function when they occur in important protein domains. For gene editing experiments ... addition to off-target activity , it is also important to consider factors that maximize cleavage of ...25408407 Hashimoto, M., & Takemoto, T. (2015). Electroporation enables the efficient mRNA delivery into the...

  14. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    ...in fewer cells being considered for the analysis. (C) A more liberal gate allows more cells to be considered...signals. For this, you’ll need to gate based on the proportion between H and A (or W and A) within the same ... just as fine (Figure 4). As H and A scale in proportion (while H and W do not; see Figure 2), single ...                                                                                                         B.    Figure 4: (A) Using the proportional relationship between FSC-H and FSC-A we can ...

  15. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ... coli." Scientific reports 5 (2015): 15096. PubMed PMID: 28060411. Thomason, Lynn C., et al. "Recombineering...merely co-opted toward that aim (i.e. sacB). Importantly, to prevent activation of toxins before the second...

  16. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    ...3):937-948. doi: 10.1016/j.ymthe.2020.11.009  Guo C, Ma X, Gao F, Guo Y. Off-target effects in CRISPR/...better to reduce off-target effects, but it’s important to not let ‘perfection’ get in the way of ‘good... score their probability of off-target events: CRISPOR, Cas-OFFinder, CCTop, and many more. While such...

  17. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    ...composed of Venus fluorescent protein, the Troponin C Ca++ binding domain, and NanoLuc® Luciferase. CalFlux...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase...share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research...(FLuc) and Renilla (RLuc) luciferase. This is important not so much for how bright you can make a reaction...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/expressing...

  18. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    ...10.1016/j.cell.2013.04.025 Zhang X, Liang P, Ding C, Zhang Z, Zhou J, Xie X, Huang R, Sun Y, Sun H, Zhang...enrich and select for cells that had successfully incorporated the desired mutation, and then derive mice from...handling embryos ex vivo, the components can be electroporated into the oviduct of pregnant females, a technique...gRNA, Cas9, and donor oligonucleotide can be electroporated into the mouse zygote (Qin et al., 2015).  ... it a single nucleotide change, insertion of a reporter gene, or replacement of the murine sequence with... microinjectionist, who has the strategically important job of delivering the payload! Last but not least...-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease. Genetics 200:423–430 . https:/...

  19. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    ...repository 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 -...findings can be predictably applied to future work. Importantly, no one should expect that every study should...of reproducibility for preclinical studies, as reported by drug companies attempting to apply the findings...and use computer based analysis techniques, an important component of reproducibility is making sure that...

  20. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...4354 . https://doi.org/10.1073/pnas.76.9.4350 Yang P-C, Mahmood T (2012) Western blot: Technique, theory,...while less protein leads to a thinner band. It is important to note, however, that you must include a loading...

  21. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ...measure this effect, known as covariance. This is the “C” that differentiates these tests from ANOVA/MANOVAs...most effective and accurate way possible it’s important to select the right test for the job (and, uh,...

  22. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ... Y, Miki T, Fujioka R, Uehara R, Tomioka A, Obuse C, Kubo M, Hiwatashi Y, Goshima G (2012) An Inducible...fluorescent reporter, directly couple silencing of your gene target to silencing of the reporter. This is...application of long or short dsRNA. However, it is important to note that naked and unmodified dsRNA elicits... Together, this excellent work highlights the importance of using orthogonal methods to confidently conclude...fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of these ...genome, whereas shRNAs are exogenously derived. Importantly, instead of the assortment of siRNAs generated...contains a target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore, ...

  23. Transferable Skills Guide: Teamwork

    Type
    Blog Post
    ... my senior year isolating pheromones from various C. elegans mutants by myself, I would have slowly gone...orchestrate the formation of high-functioning-but-temporary teams that have a specific purpose and finite ...year, or a few months, or even a few weeks. The important thing to remember is that every team should have...when I do come across one. Also, remember, one important feature that all projects have in common - and... What does a good team need? Besides that all-important sense of purpose, there are many more things necessary... function is certainly necessary, but equally important is making sure all team members understand what...their responsibilities are. That’s a subtle but important distinction. If I invite someone to be on a project...

  24. Which Fluorescence Microscopy Techniques is Best for Me?

    Type
    Blog Post
    ...920-931 (2005). PubMed PMID: 16299477. 3. Eggeling C, Willig KI, Sahl SJ, Hell SW. Lens-based fluorescence...Harvard University. No matter whether you are a sports photographer at the Super Bowl, a medical technologist...the first fluorescence microscopy technique to incorporate optical sectioning. Optical sectioning refers...their fast movements. Depending on the sample, a temporal resolution on the millisecond scale may be needed...plane of excitation light that is thinner than the portion of the sample that is in focus. This technique ...resolution due to the lack of a requirement for high temporal imaging speeds. Standard fluorescence widefield...sectioning, imaging over large distances and high temporal resolution. Traditionally, this type of sample...

  25. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals an essential role... methods allowed immunologists to develop a spatiotemporal view of an immune response, but the study of...transgenic mice expressing fluorescent protein reporter constructs in the immune lineage. By specifically...than the standard Ti:Sapphire laser. It is also important to finely tune the excitation wavelength as it...using 2-photon microscopy as this hasn't been well reported for many fluorescent proteins. Additional fluorescent...

  26. CRISPR Guide

    Type
    Guide
    .... A., Amrani, N., Peng, C., Kramme, C., Savic, N., Pacesa, M., Rodríguez, T. C., Stan, T., Tysinger, E...Dalvai, M., Loehr, J., Jacquet, K., Huard, C. C., Roques, C., Herst, P., Côté, J., & Doyon, Y. (2015)....Heussler, G. E., Hearne, C. C., Qu, J., Inclan, Y. F., Hawkins, J. S., Lu, C. H. S., Silvis, M. R., Harden...this strategy yields C to A base edits, whereas in mammalian cells, it produces C to G edits. Adenine ... Cas3 must be paired with the Cascade ( C RISPR- as sociated c omplex for a ntiviral de fense) complex...and Cas12a (also known as Cpf1) Cascade C RISPR- as sociated c omplex for a ntiviral de fense; a complex..., S., Bietz, A., Waldhauer, M. C., Börner, K., Fakhiri, J., Schmelas, C., Dietz, L., Grimm, D., Correia...

  27. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library...sub-cellular localization signals, plant markers, and reporters (Engler et al., 2014). This kit is a great starter...upon (its ‘basics’ MoClo plant kit buddy). It is important to understand your experimental needs before choosing...into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This allows for the multiplexing of sgRNAs...basic biology of and gene regulation in the opportunistic pathogen Acinetobacter baumannii (Brychcy et...used successfully for protein localization and transport studies in HEK cells, recombinant protein purification...Multi-Kingdom Golden Gate Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media...

  28. Molecular Biology Reference

    Type
    Guide
    ... T H A, C, or T K G or T M A or C N A, T, C, or G R A or G S C or G V A, C, or G W A or T Y C or T Amino...Thymine (T), Cytosine (C) and Guanine (G). In the double helix A always pairs with T and C always pairs with...Adenine C Cytosine G Guanine T Thymine U Uracil Single Letter Code: Ambiguous bases Nucleobase B C, G, or...thymine, cytosine, and guanine (abbreviated to A, T, C, and G respectively) that are organized into a double...1000X stock solutions and storing aliquots at -20°C. To use, dilute your antibiotic into your LB medium...complementary, strand. Specifically, A pairs with T and C pairs with G. During replication, DNA unwinds and ...growing DNA strand in addition to the normal A, T, C, or G nucleotide. The 4 standard bases are tagged ...

  29. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing oligos C.3 Digesting...Virus may be stored at 4°C for a few days, but should be frozen at -20°C or -80°C for long-term storage...., R is a purine (A,G), and Y is a pyrimidine (C,U). G-C content should be 36-52%. Sense 3’ end should ...annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning vector from step C.3 (If you were unable ...Store at 4°C. m. Add 5 mL of fresh media containing antibiotics to the cells and incubate at 37°C, 5% CO ...at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately 80-90% confluent. c. Dilute...solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several years...

  30. Kit Free RNA Extraction

    Type
    Protocol
    ...before you start. Homogenizer Vortexer -20°C freezer -80°C freezer Reagents Solution D (for Protocol Option...instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight. Centrifuge for 20...centrifuge the sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate... lysate. Pro-Tip Having your samples spin at 4°C helps reduce RNA degradation. If you don’t have access...Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium Chloride (Option B): LiCl selectively...extracted aqueous layer to 2.5 M. Incubate at -20°C for 1 hour. Pro-Tips If you anticipate your RNA yield...
Showing: 331 - 360 of 395 results