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Showing: 181 - 200 of 342 results
  1. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

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    Blog Post
    ...in our hands, is both cost-effective and highly active. After csRNP complex formation, a single-stranded...efficiently and serves as a good control for both the activity of CRISPR reagents and ICC antibody specificity...of the crRNA (aim for 40-60% GC content) crRNA activity Local chromatin structure (although we could successfully...turnaround time 5-7 days); because the tracrRNA is the same in all csRNP experiments, it can be ordered in bulk...
  2. Addgene's Website Update

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    Blog Post
    ...new deposit materials bar New menu Most common activities can be done straight from the new search bar,...Materials”. The “Search Repository” function is our same search bar. And if you’re in the mood to browse ...
  3. Rett Syndrome: A History of Research and Therapeutic Outlooks

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    Blog Post
    ...transcriptional repression, activity dependent de-repression, chromatin remodeling, gene activation, and more. Loss...gene replacement therapy, gene editing, MECP2 reactivation, RNA editing, RNA trans-splicing, and protein...They introduced a mouse MECP2 guide RNA and a hyperactive version of the human RNA base editing enzyme ...https://doi.org/10.3389/fgene.2019.00625 McGraw CM, Samaco RC, Zoghbi HY (2011) Adult Neural Function Requires...
  4. Plasmids 101: E. coli Strains for Protein Expression

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    Blog Post
    ...are insoluble, act on DNA, or are enzymatically active. For this reason, recombinant proteins are typically...* Streptomycin and Tetracycline Contains highly active thioredoxin reductase and glutathione reductase...proteins. hsdSB (rB- mB-): These strains have an inactivated native restriction/methylation system. This means...-deficient; allows for cloning and expression in same strain F- recA1 hsdR(rK12- mK12+) (DE3) (RifR) ...plasmids utilize inducible promoters, which are 'inactive' until an inducer such as IPTG is added to the...
  5. Easing the Protein Purification Process with pCri

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    Blog Post
    ...purification can be one of the most stressful lab activities. Working with proteins requires a substantial...N- and C-terminal. Each set of plasmids uses the same restriction enzymes for cloning (see schematic below...one need to remove the tag post-purification. A sample of pCri constructs: pCri-1a, -4a and -9a. 6xHis...
  6. Which Fluorescence Microscopy Techniques is Best for Me?

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    Blog Post
    ...have allowed for real-time monitoring of neuronal activity across substantial 3D volumes. In addition to ...Weijian, and Rafael Yuste. In vivo imaging of neural activity. Nature Methods 14.4 (2017): 349-359. PubMed PMID...due to the refractive index mismatch between the glass and the water-like buffer the sample is immersed...microscope to use for a new sample I always start by asking two questions: 1) Is the sample dynamic or static? ...? 2) Is the sample thin (< 15 μm) or thick? These questions will place each sample into one of four categories...capture in these types of samples is their fast movements. Depending on the sample, a temporal resolution... fluorophores higher in the sample. TIRF is also very gentle on the sample as the majority of the laser...
  7. Technique: Probe Phage Genomes for Host Binding Proteins

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    Blog Post
    ...genome to be probed for proteins with host-binding activity without the need for genome sequencing or annotation...nitrocellulose for subsequent probing for cell-binding activity.  First, cut out 20 Petri dish-sized circles of...at the same location that their host colonies grew. If your cells of interest stick to the same location...
  8. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...population in a procedure termed fluorescence activated cell sorting or FACS (2). By analyzing single ..., Sweet RG, Herzenberg LA (1972) Fluorescence Activated Cell Sorting. Review of Scientific Instruments...was raised in the same host species, of the same Ig subclass, and conjugated to the same fluorochrome as...ensure that samples are single cell suspensions. For cells that tend to aggregate, pass the sample through...using an amine reactive dye, such as LIVE/DEAD stain. Like DNA binding dyes, amine reactive dyes can only...single-stained samples for each fluorochrome in the experiment. Single-stained samples must be as bright...experimental sample. In addition, you should include a panel of “fluorescence-minus one” controls, samples stained...
  9. Plasmids 101: Common Lab E. coli Strains

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    Blog Post
    ...  LacZ β-galactosidase activity abolished   lacY Lactose permease inactivated, lactose cannot be taken...increases plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability, reduced...convoluted due to researchers sharing and renaming samples throughout history. It was likely isolated in 1918...taken up by cell mcrA, mcrBC Inactivation of pathway that cleaves methylated cytosine DNA Allows for ... foreign (methylated) DNA mrr, Δ(mcrC-mrr) Inactivation of pathway that cleaves methylated adenine or...source leuB β-isopropyl malate dehydrogenase inactivated Requires exogenous leucine source for growth ...proAB lacIqZΔM15 Tn10(TetR Amy CmR)]  Note: Inactivating mutations in specific genes are signified with...
  10. Important Considerations When Using AAVs

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    Blog Post
    ...chemogenetics (Designer Receptors Exclusively Activated By Designer Drugs, DREADDs) and optogenetics as...use of DREADDs (Designer Receptors Exclusively Activated By Designer Drugs) to determine the role of serotonin...chemogenetic receptors delivered by AAV within the same cell types. Not only can we compare these methods...finger nucleases, and many others (12). The most attractive potential of AAVs is their ability to be used...McCarty, D. M., Paul E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated...PMC190186. 9. Grieger, Joshua C., and Richard J. Samulski. "Packaging capacity of adeno-associated virus...
  11. Live and Let Dye: Self-Labeling Protein Tags

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    Blog Post
    ...enzyme that has been mutated to block its catalytic activity and instead become trapped in a covalent bond ...brightness, stability, or other properties is an active area of research. If size is a concern for your...sequentially adding different ligands to the same sample to label sub-populations of your protein of interest...probably find lots of different plasmids containing the same protein of interest tagged with different fusion...affinity purification tags, another version with that same red fluorescent protein somebody made because they...ligands (Gautier et al., 2008). Although based on the same original enzyme, SNAP-tag and CLIP-tag show good...specificity for their own ligands with minimal cross-reactivity. Other strategies include using engineered ligases...
  12. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    ...proteins in tissues, cells, and biofluids. Recently, Sam Myers, a Scientist in the group, developed a CRISPR-based...and RNA sequencing of complex heterogeneous RNA samples. Furthermore, such high processivity enzymes and...specific genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered peroxidase...Using these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9) fused to APEX2 is expressed from...
  13. RUBY-Red Siliques

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    Blog Post
    ...A) Betacyanin is produced from tyrosine by the activity of 3 enzymes and a spontaneous step. (B) RUBY ...GUS staining or are not suited to studying large samples. RUBY Red But recently, something new has been ...consisting of these three enzymes expressed under the same promoter with self-cleaving 2A site linkages and...transformation next to an untransformed seed from the same plant beginning to germinate. (Top right, second...
  14. Stepping into a Virtual Internship: My Experience at Addgene

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    Blog Post
    ...invited to speak to other Addgenies. I could take an active role in the company and not just feel like an additional... I pondered on the ways that I wouldn't have the same experience as actually being in the office. However...
  15. Antibodies 101: ChIP

    Type
    Blog Post
    ...downstream analysis focuses on the DNA your protein is actively interacting with, reducing background information...you’re working with a tissue sample, you’ll need to homogenize your sample before moving on to the next...Homogenization for a tissue sample can also need significant optimization for the sample type and size.) It can...process is very similar for both, though tissue samples do require an extra homogenization step. For this...to focus on the ChIP process and not the upsteam sample collection process or the downstream analysis process...itself begins at...  Crosslinking Once you have your sample, the first step is to ensure your protein:DNA complexes...likely to remain attached to the DNA throughout the sample processing and therefore will require crosslinking...
Showing: 181 - 200 of 342 results