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We narrowed to 988 results for: Ran

Showing: 261 - 280 of 988 results

  1. Special Delivery: Fluorophore Targeting for FRET Studies

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    Blog Post
    ...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs... minimum energy transfer values you can expect, thereby establishing the dynamic range of your FRET assay...what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two...first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second fluorophore...structural changes in the ribosome during protein translation (Ermolenko et al., 2007). All that is required...develop a successful FRET-based assay since energy transfer only occurs when donor and acceptor are close ...Å away from the donor doesn’t result in energy transfer and thus, your specific FRET signal is unaffected...

  2. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...project forward, transductions should not be rushed into. Before you start any transduction, you should always...Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used... positive for vector encoded transgene expression following transduction with serial dilutions of a viral...resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions of a ... your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time ...assaying for expression of a gene carried on the transfer plasmid or quantifying the number of viral copies...gag, WPRE, antibiotic resistance-genes, or the transgene itself. Many researchers prefer to design primers...

  3. Plasmids 101: Inducible Promoters

    Type
    Blog Post
    ... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...lag time due to the time needed for transcription/translation. Many thanks to Nicole Zurcher who contributed... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive and cannot bind the tetracycline...

  4. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ...packages have spent days to weeks in transit and some have been transported across oceans and continents. Each... treated to easily take in plasmids (transformation). Transformed bacteria are then grown up on antibiotics...received is immediately transformed and stored. Some samples need to be transformed with a micropulser using... performs an average of 140 transformations. The lab typically transforms newly deposited samples from...delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted...long-term storage in our facilities, every sample is transferred to a 2D barcoded tube (see next section) and ...make a new tube from a replicate sample. The transformation After the DNA has been received and stored,...

  5. Understanding GPCR Signaling Using TRUPATH

    Type
    Blog Post
    ...-visual G protein signal transducers (collectively known as the transducerome) that can be activated downstream...Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which...non-visual G protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing...including smell, taste, sight, inflammation, and neurotransmission. But with approximately 950 different human...involved in sight (Olsen RHJ et al., 2020). A transducer complex consists of a trimer of three subunits...such a tool is being able to identify specific transducer complexes that are activated in response to a...fluorophore. The difference for BRET is that the energy transfer is between a luminescent donor and a fluorescent...

  6. RUBY-Red Siliques

    Type
    Blog Post
    ...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...For example, with CRISPR-Cas9 gene editing, transformation may be identified by resistance to an antibiotic...antibiotic. Successful editing of transformants then typically occurs in T0 plants (from callus) or T1 plants...contaminate it. I have used it for selecting transformation events and am currently planning to use it ...

  7. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...their position relative to a transcription start site. In STARR-seq, randomly sheared genomic libraries ...of these random stretches of DNA contain an enhancer, it will activate its own transcription from the ...alone. Molecular processes from signal transduction to transport rely on protein-protein interactions and...an alternative method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests...single gene. The gRNA expressing cells were then transfected with a Cas9-expressing plasmid to generate a ... Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC...RAS signaling pathway and includes 180 unique transcripts. Each gene was chosen to represent the primary...

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

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    Blog Post
    ...light-dependent activity to transcription, they fused BldD to the transcriptional activators p65 and VP64 ...readily found at Addgene. Shining light on transcriptional activation using dCas9 Initial photoactivatable...early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato’s lab at ...photolyase homology region (CRY2PHR) fused to a transcriptional activator domain (VP64 or p65). Upon expression...the activator into position to activate gene transcription. The researchers tested a variety of combinations...CRY2FL-VP64, a fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in...(Nihongaki, et al., 2015) resulting in a double strand break (DSB) that can be repaired by either non-...

  9. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...specific titer, you can better plan your transductions and use a range of reasonable MOIs. 2. Create a monoclonal...generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected...generate DNA duplexes with one strand from the edited cells and one strand from the control cells, which.... The appearance of the 428 bp band indicates edits in the BRAF gene in the BRAF-gRNA transduced cells...generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting...whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing lentivirus at the indicated...

  10. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ...can be motifs within transmembrane proteins, such as receptors, or post-translational modifications on those...diffuse through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones...Depending on the application, they can be membrane-permeant or membrane-impermeant, and the cells of interest...viability dyes are membrane-impermeant and can only enter cells with compromised plasma membranes — i.e., dead...conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility ...epitope-targeting antibody can originate from a range of species; for example, rabbit or mouse. In a second...

  11. Plasmids 101: Protein Expression

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    Blog Post
    ...DNA must first be transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into...consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe, translate...expression post-transfection. Mammalian systems can express proteins both transiently or through stable...expression vector that is then transformed into a bacterial cell. Transformed cells propagate, are induced...vitro using purified components of the transcription and translation machinery. These include ribosomes, ...proteins are then modified via an array of post-translational modifications including protein folding, formation...proteins from mRNA and the addition of post-translational modifications Learn more molecular biology ...

  12. Hot Plasmids: Spring 2025

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    Blog Post
    ...al., 2025). In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses homology ...fused to a shortened R2Tocc transposon protein, and can "retarget" the transposon to new loci in the genome...genome. The modified transposon RNA has untranslated regions (U, blue) on the outside of the homology arms...-associated membrane protein 5 (LAMP5) — as well as cholinergic (choline acetyltransferase, ChAT) neurons...is one of the most important and abundant neurotransmitters, but it is challenging to monitor because ...rapidly taken up by postsynaptic receptors and transporters. The intensity-based Glutamate-Sensing Fluorescent...deposited versions with both PDGFR and NGR fusions for membrane display, they recommend the NGR vectors for most...

  13. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

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    Blog Post
    ...strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Non-random integration...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...Danio rerio) have been used since the 1930’s in a range of biological studies, including investigations ... to produce abundant, externally fertilized, transparent progeny (Figure 2). These features give it a ...individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...mutants In the 1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants...

  14. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...expression transposases leading to increased integration frequency. (C) Increasing rounds of transfer on transposition-inducing... engineering single genome edits but the double strand breaks it introduces makes it difficult to perform...chromosomal integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these... This would result in the lack of recovered transformants. Our speculation motivated us to update the ... sequence in pEcgRNA. Next both plasmids are transformed in E. coli with the donor DNA for editing to ...

  15. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    ... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...is fused to CBP, an histone acetyltransferase domain capable of rearranging chromatin structure. This method...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...gene regulation. This can be used to activate transcription during either initiation or elongation, depending...uses specially engineered sgRNAs to increase transcription. This is done through creating a dCas9/VP64 ...

  16. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    ...previously require a preliminary double stranding step. The double stranding step was assumed to be necessary...genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  ...extraction, the [+] and [-] strands might naturally associate forming a double-stranded species that could be...extract is a heterogeneous mix of single-stranded and double-stranded species. While not all of the molecules...extract associate with complementary strands, enough double-stranded species exist in the mix to serve as...viral genome We hypothesized that this double stranding step might not actually be necessary. Since the...the AAV genome exists as [+] and [-] strands that are packaged at the same frequency, we reasoned that...

  17. Multicolor Animals: Using Fluorescent Proteins to Understand Single Cell Behavior

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    Blog Post
    ...), YFP (yellow), and membrane-tethered CFP (cyan). Brainbow 1.1 adds OFP (orange) (Livet et al., 2007)... mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic...labeling technique involves the introduction of a transgene construct containing fluorescent proteins (XFP...Because each cell can have multiple copies of the transgene that will recombine independently, cells may acquire...respectively, cells will fluoresce either red or orange until recombination occurs. Following the development...incorporates the highly photostable fluorophores mOrange2, EGFP, and mKate2 (red) to solve the problematically...non-fluorescent form of YFP as the default state of the transgene such that recombination is necessary to excise...

  18. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    ...format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation...flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period... regardless of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists...interactions. In later years, improvements to yeast transformation protocols also helped establish the yeast two-hybrid...reporters and some that repress instead of activate transcription. The HIS3 reporter system (e.g. the Gateway-...even possible to have Y2H systems that avoid transcriptional reporters altogether; In the split-luciferase...group of prey fusions can either be generated using random genomic fragments from the organism of interest...

  19. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...cells in vitro. I transduced the cultured cells with a virus that encodes a transgenic protein for the ...having a separately expressed transduction reporter is that not every transduced cell will necessarily manage...the transgenic protein. We don't know yet how abundantly CD45 will be expressed on the transduced cells...panel to analyze transduced HEK 293T cells. Here, we looked at both the transduction efficiency and the...emission. The visible color spectrum is located in a range of wavelengths of about 380–700 nm. Photons of lower...electrons of a fluorophore, they can do so at a range of wavelengths, rather than at just one defined ...Likewise, the emitted photons also appear within a range of wavelengths. For that reason, we speak of excitation...

  20. Viral Vectors 101: AAV Serotypes and Tissue Tropism

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    Blog Post
    ...  AAV8 AAV8 has high transduction efficiency in multiple tissues, rapid transgene expression, and can ...undergo retrograde transport, traveling up the axon to the cell body and driving transgene expression (Tervo... S. (2013). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5...vitreous. Science Translational Medicine, 5(189), 189ra76. https://doi.org/10.1126/scitranslmed.3005708 Goertsen...N-linked sialic acids1 AAV receptor2   AAV2 Heparan sulfate proteoglycan (HSPG)3 AAV receptor (not ...good choice for targeting neurons, with high transduction frequencies in the central nervous system (CNS...Asokan, et al., 2006). AAV1 has also been found to transduce skeletal muscle, cardiac, and retinal cells (Issa...
Showing: 261 - 280 of 988 results