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We narrowed to 809 results for: ran

Showing: 361 - 380 of 809 results

  1. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    Published
    Dec. 1, 2016, 3:30 p.m.
    ... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...selection marker HSV-tk is used to select against random recombinants.   Design your targeting construct...selecting for cells in which the construct has randomly integrated into the genome, a negative selection...step will kill off many of the cells that have randomly integrated all or large portions of the plasmid...negative selection marker selects against improper, random recombination into a different locus. Correctly...contain the negative selection marker, but cells with random recombination may incorporate the negative selection...and animal models, their efficiency is very low, ranging from undetectable to 0.1%. In contrast, new genome...

  2. Antibodies 101: Producing Recombinant Antibodies

    Type
    Blog Post
    Published
    Aug. 13, 2024, 1:15 p.m.
    ...mammalian cells can perform human-like post-translational modifications that bacterial and most yeast ...be produced through a single or dual-plasmid transfection, depending on whether the heavy and light chain...they’ve been separated onto two. You’ll first transfect your cells with your plasmid(s), and then wait... from dropping the tube they are contained in (Randolph et. al., 2018). At Addgene, we ensure the efficacy... Bisson L, Gervais C, Grazzini E, Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells...A. Pathak, Christopher J. Roberts, Prasad S. Sarangapani, Protein aggregation, particle formation, characterization..., https://doi.org/10.1016/j.cocis.2014.10.002. Randolph TW, Schiltz E, Sederstrom D, Steinmann D, Mozziconacci...

  3. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    Published
    July 31, 2020, 12:30 p.m.
    ...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target...deaminase ADAR2DD to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the ...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note...therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging the construct...

  4. Capturing a Moment with Marc Zimmer’s “The State of Science”

    Type
    Blog Post
    Published
    July 30, 2020, 1:15 p.m.
    ... that may have warranted their own chapters, and certainly chapters that could warrant their own books..., and he wisely makes no such attempt. He is transparent about his own biases--many of which will be shared...non-scientists, Zimmer’s observations will likely range from mildly surprising to genuinely shocking. Mainstream...he describes the now infamous tale of Rosalind Franklin and her essential-but-unlauded role in the identification...within this section. The GFP story has its own Franklin in Doug Prasher, who originally isolated the GFP...science produced by the quieter, more reserved Franklins and Prashers will find it increasingly difficult...

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

    Type
    Blog Post
    Published
    May 2, 2019, 12:41 p.m.
    ...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...Phenotype thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific...evolution because electric organs arose in different branches of the evolutionary tree at least 6-7 times and...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... a system using novel vesicles to efficiently transform these cells which don’t like foreign DNA at all...One can inject plasmid DNA in the eggs to get transgenic animals. They might be immortal. Manihot esculenta...ReMOT control system (Receptor-Mediated Ovary Transduction of Cargo)  delivers Cas9 RNP to the arthropod...

  6. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    Published
    Feb. 10, 2015, 3:06 p.m.
    ...specialized strains are available to confer greater transcriptional control, assist with proper protein folding...harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...pLemo (CamR) Expression of toxic, insoluble, or membrane proteins  T7 Express   IPTG-inducible expression...addition of IPTG. T7 RNAP is then available to transcribe the gene of interest from a T7 promoter on the... the expression plasmid in order to repress transcription from a hybrid promoter. Although the DE3/T7 ...

  7. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    Published
    Dec. 20, 2017, 2:33 p.m.
    ...the non-profit sector are transferrable to industry such as technology transfer, communications, medical... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a...and therefore the academic H-1B visa cannot be transferred to an industry position. If you cannot get an... facility management, collaboration management, grant administration, clinical operations, and so on. ...include evidence such as proof of US government grants that the researcher is working under in the lab...new accomplishments to add. Obtain copies and translations of documents that are not in English. Create...    Additional Resources on the Addgene Blog Transferable Skills Guide 6 Steps to Submitting a Resume ...

  8. March for Science

    Type
    Blog Post
    Published
    April 21, 2017, 2:30 p.m.
    ...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...Post: Trump administration tells EPA to freeze all grants, contracts The Guardian: Trump to scrap Nasa climate... Scientists Find a Voice at Massive Rally for Immigrants National Science Foundation: Universities Report...

  9. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends...the adenosine of the GATC recognition site. Transformation: After the PCR reaction, no ligation is required...required since the E. coli you transform your PCR products into will efficiently patch up the DNA. The ...parental plasmid provides a mean for selecting for transformants which have taken up the mutagenized plasmid....

  10. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ...therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using ...histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. For a targeted...dCas9-VP64 fusion commonly used to activate transcription, but with two additional activation domains ...domains (MS2 and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target ...functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-1 cleavage allows T-cells...rates of off-target cleavage. In both cases, translating the in vitro work into an animal model comes ...cleavage. Despite the potential difficulties in translating these findings into a therapy, these papers present...

  11. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...of different fluorescent protein are available (Cranfill et al., 2016 and FPbase.org). A number of variants... organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is big GFP is a ...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...clearly the case when proteins undergo post-translational modification with fatty acyl chains. For instance...Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells." Science 296.5569 ...28360214. PubMed Central PMCID: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of ...PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent proteins to study mRNA trafficking...

  12. Seeing Red: Simple GFP Photoconversion

    Type
    Blog Post
    Published
    Aug. 4, 2015, 2:30 p.m.
    ...Verkhusha VV. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nat Methods. 2011 Jul 31;...proteins Newly developed photoconvertible FPs like PsMOrange and mEos add another layer of complexity to imaging...GFP spreads through the cytosol via cytoplasmic strands. Image from Sattarzadeh et al., Sci Rep. 2015 CC...most useful for labs that already have created a transgenic GFP-containing organism,” says Hanson, “using...unexpectedly photoconvertible GFP proteins to make a new transgenic line could avoid problems in localization that...

  13. Easing the Protein Purification Process with pCri

    Type
    Blog Post
    Published
    June 19, 2015, 3:08 p.m.
    ...contains the MISTIC (membrane-integrating sequence for translation of integral-membrane protein constructs...glycosylate proteins and may recapitulate the post-translational modifications added to proteins in other eukaryotes...includes a special feature for difficult-to-purify membrane proteins. pCri vector pCri-13a, designed for use...protein tag. MISTIC drags tagged proteins into the membrane and may improve expression of proteins that natively...

  14. What's New in CRISPR - September 2019

    Type
    Blog Post
    Published
    Sept. 10, 2019, 12:56 p.m.
    ...proteins, or transcriptional regulators. In this system, Cas9 generates a double-stranded DNA break at...are complementary to either side of the double-stranded break in the genomic loci of interest. As the ... Find the CasX plasmids Drug inducible transcriptional activation and genome editing Yu Wang's lab...developed drug inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9...library contains ~93,000 unique gRNAs that target random positions along the E. coli MG1655 genome with ...

  15. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...orthogonal attB-attP pairs per recombinase may be arranged on a register (Fig. 1C below). If attB-attP pairs...approach is to interrogate state with qPCR. The re-arrangement of DNA segments in each state of a register enables...sensors do not necessarily have to be based on transcriptional regulation, as long as they can control recombinase...recombinase activity. For example, post-transcriptional recombinase activation via RNA toehold riboregulation...expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register to record all permuted... of a set of chemicals. Specific examples of arrangements that should be able to record all permuted substrings... this is to prevent interference between the transcribed gene and its 5’ UTR. Various self-cleaving hammerhead...

  16. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    ... charged ions to rapidly pass through the cell membrane and initiate an action potential - a very useful...and close when exposed to light and change the membrane potential. OptoXRs are modified GPCRs. When exposed...downstream signaling pathways that can change both the membrane potential and a host of other cellular functions...activity upon exposure to light within a specific range of wavelengths. Excitatory channels allow positively...Photoswitches are great for teasing out important branch points along a signaling cascade that may go awry...optimal wavelength to use but can be activated by a range of wavelengths. While this may give you a little...Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proceedings of the National Academy of...

  17. Plasmids 101: Gene, enhancer, and promoter trapping

    Type
    Blog Post
    Published
    May 2, 2023, 1 p.m.
    ... gene, enhancer, and promoter – all rely on the random integration of a reporter construct in close proximity...integrate within an exon of a gene to generate a transcriptional fusion. This may disrupt the endogenous function...spliced into the gene product resulting in a transcriptional fusion, much like promoter traps. While this...likely disrupt the gene it monitors due to transcriptional fusion.   Examples of promoter, enhancer...monitored genes and mutants - viral integration, transposon, and many more! Check out Addgene’s catalog to...resources on the Addgene blog Plasmids 101: Using Transposons in the Lab Plasmids 101: Screens vs. Selections...

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

    Type
    Blog Post
    Published
    May 21, 2020, 1:15 p.m.
    ...visualize individual/non-overlapping cells. While transgenic animals are commonly used to specifically manipulate... to trigger recombination and expression of a transgene (Lin et al., 2018). However, this system requires...tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site. GAL4 expressed in the fly...site to cleave and inactivate any read-through transcripts of the effector gene. Effector genes, any gene...across cell types and organisms by replacing the transcription factor binding site and effector in the construct... the SPARC system to use other experimental transcription activation systems, such as LexA/p65 or mCD8...

  19. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    Published
    Aug. 19, 2014, 3:39 p.m.
    ...). If you can get away with generating transiently transfected cells, you will not need a mammalian selection...mammalian selection cassette to screen for positive transfected cells. The addition of an in-frame fluorescent...create a new strain with a disrupted gene by transforming wild-type yeast with an integrating vector expressing...majority of fly vectors are based on p-element, random or targeted, integration into the fly genome and...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...

  20. Bright Monomeric Fluorescent Proteins: mNeonGreen, mTFP1, and mWasabi

    Type
    Blog Post
    Published
    April 25, 2019, 3:01 p.m.
    ...protein (LanYFP) from the marine invertebrate Branchiostoma lanceolatum in 2012. Using structural modeling...mNeonGreen can be used as a fusion tag for a wide range of applications from traditional imaging to FRET...Alberta and University of Oregon created mTFP1 by transforming E. coli with a gene library of approximately...mTFP1 can be used as a FRET donor to mCitrine and mOrange or for live cell imaging of fusions and FRET reporters...particularly with fluorophores that excite in the violet range. For example, mWasabi can be used with Sapphire ...monomeric green fluorescent protein derived from Branchiostoma lanceolatum." Nature methods 10.5 (2013): 407...
Showing: 361 - 380 of 809 results