We narrowed to 1,054 results for: tra

... NOTE: All zinc finger arrays listed show transcriptional activation activity of three-fold or greater...

... Cre dependent 5 Tim Brown and HHMI-JRC Tool Translation Team 114471 pAAV-Ef1a-fDIO mCherry EF1a mCherry...

...for transfecting mammalian cells using linear polyethylenimine. Transfections allow for transient expression...RT. Carefully transfer the transfection mix to your HEK293T cells. Add the transfection mix dropwise, ... viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace with...protocol to transfect mammalian cells with your plasmid of interest. Protocols... Protocols General Transfection General...General Transfection You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...adapted for different cell lines and different transfection reagents. Workflow Timeline Day 0: Seed HEK293T...

...Introduction Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria...modifications to create bacterial strains that can be more easily transformed and that will help to maintain...prepared for optimal transformation efficiencies upon thawing. For the highest transformation efficiency, we...time when carrying out transformations? If you are not concerned with transformation efficiency (such as ... 20-30 mins) I get very few if any transformants when transforming large plasmids (>10 kb) or BACs, what...Learn how to transform E. coli with your plasmid of interest. Protocols... Protocols Bacterial Transformation Bacterial...

... Protocols ddPCR Titration of AAV Vectors ddPCR Titration of AAV Vectors You...for purified AAV titration. Protocols...You may also like... AAV Titration by qPCR AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular...samples. ITR Master Mix Volume 9X Volume Final Concentration 2X ddPCR Supermix for Probes, no dUTP 10 µL ...the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator....while slowly and gently aspirating the droplets. Transfer the droplets to a prechilled PCR plate. Pro-Tip... 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft...

...Colony Formation Titration Assay Lentivirus Fluorescence Titration Assay AAV Titration by qPCR Addgene ... Protocols ddPCR Titration of Lentivirus Vectors ddPCR Titration of Lentivirus...droplets (blue/green). The concentration of RRE positive droplets in the untransduced control should be close...serially diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and ...+06 7 Untransduced 1 6.4 17940 0.0007134894091 1.43E+02 Table: Example dilutions and titrations Reference...lentivirus vector titration. Protocols...titer range of 1E+05–1E+09 TU/mL, where TU is transducing units and generally represents the number of ...

... to proceed with transfection. Transfect the flask containing 108 mL cells: Transfer 6 mL of BCD TFX into...General Transfection Protocol Recombinant Antibody Purification Protocol Introduction Transfections allow...using Polyethylenimine Max as a transfection reagent. After transfection and expression, the recombinant...Section 2: Transfection Check the cell density and viability: Using a 5 mL pipette, transfer 0.5 mL of ...feeding schedule: Thursday: Transfect cells. Friday (24 h post-transfection): Add 4% BCD Feed. Monday (...post-transfection): Add valproic acid to 3.75 mM, add 4% BCD Feed. Tuesday (120 h post-transfection): Add...Wednesday (144 h post-transfection): Add 4% BCD Feed. Thursday (168 h post-transfection): Harvest antibody...

... Protocols RNA Extraction Without a Kit RNA Extraction Without a Kit...effectively lyse open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4....5 M LiCl solution to bring the concentration of LiCl in the extracted aqueous layer to 2.5 M. Incubate...perform RNA extraction without a kit. Protocols...Reference Introduction This protocol describes how to extract RNA from tissues or cells without a kit. The general...include homogenization/lysis of cells or tissues, extraction of RNA, precipitation, and resuspension. This...

...Iodixanol Gradient Ultracentrifugation AAV Purification by Iodixanol Gradient Ultracentrifugation You may also...medicine, iodixanol can be used as an intravenous isomolar contrast agent. In research, iodixanol is used...and concentration Note: Both steps could be completed in one (long) day. Equipment Ultracentrifuge T70i...the column. We recommend concentrating to a minimum of 500 µL. If the concentrate volume is less than 500...Follow this protocol using Iodixanol gradient ultracentrifugation to get pure adeno-associated virus (AAV) ... pooled for dialysis. Pool clean fractions. Concentration and buffer exchange: Stocks of Poloxamer 188...Data Figure 1: Iodixanol gradient before ultracentrifugation. The steps are clearly distinguishable thanks...

... Protocols AAV Titration by qPCR Using SYBR Green Technology AAV Titration by qPCR...AAV Titration protocol. Protocols... Posts Mol Bio Protocols Introduction The AAV Titration protocol can be used to determine the number of...Size in bp of Addgene plasmid #59462 : 6208 bp Concentration of Addgene plasmid #59462 : 1.07 μg/μL Molecular...again but store at 4 °C and use within 1 week. Keep track of the Ct value for each standard over time. They...over from the production process (DNase does not penetrate the virion). 5 μL sample + 39 μL H 2 O + 5 μL ...sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do NOT treat your plasmid ...

...Iodixanol Gradient Ultracentrifugation AAV Titration by ddPCR AAV Titration by qPCR Using SYBR Green Technology...double-stranded DNA genome. Adeno-associated viruses (AAV), however, are small single-stranded DNA viruses...plasmids are needed: Transfer plasmid (also known as cis plasmid) — containing the transgene of interest between...Production Cloning Cloning your transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with...recombination deficient bacterial strains, such as NEB Stable cells. These strains reduce the frequency of homologous...three production plasmids (transfer, packaging, and helper plasmid) are transfected into cells which then ...different serotype(s) for transduction of a given organ. Tissue Serotype Central nervous system AAV1, AAV2...

...deletion is transferred to the 5’ LTR after one round of reverse transcription, inhibiting transcription of the...centrifuged to concentrate. You can use crude or concentrated viral particles to transduce the cells of ...cannot be used in direct transfections for transient expression of a transgene. For more detailed protocols...cis Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency...packaging plasmids. tat in trans Trans-activator; binds TAR to activate transcription from the LTR promoter...crossover event between the transfer plasmid and genes provided in trans . Transduction The process of artificially...for third generation) plasmids: Transfer plasmid — contains transgene, sgRNA, or shRNA of interest flanked...

...may be a single-stranded oligonucleotide, double-stranded linear DNA, or a double-stranded DNA (dsDNA) plasmid...including transposons, integrases, and recombinases, with Cas enzymes. CRISPR Transposases Transposon systems... HNH, cleave opposite strands of the target DNA. This results in a double-strand break (DSB) within the...discovered. Browse Plasmids: Single-Strand Break (Nick) , Double-Strand Break (Cut) Increasing Cas9 PAM flexibility...incorporated into the DNA by reverse transcription. The edited DNA strand displaces the unedited 5’ flap, ...Tn7-like transposases found in diverse species of microbes. However, synthetic dCas9-transposase fusions...recruits the transposase proteins to the target location in the genome, where the transposase then carries...

...to form one: Shuttle/transfer plasmid (e.g. pAdTrack) — containing the transgene of interest and both ... the transduced cells, transgene expression delivered through rAdV vectors is normally transient and short-lived...double-stranded DNA genome. Adeno-associated viruses (AAV), however, are small single-stranded DNA viruses... and late adenoviral transcript regions between the two ITRs with the transgenes of interest. The size...Shuttle plasmid (also known as transfer plasmid) — containing the transgene of interest, the ITRs, and homology...possibility that recombination between the transfected adenoviral transfer plasmid and the E1 region integrated...Production Cloning Cloning your transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with...

...sense or coding strand of the transcribed gene (Figure 1). The coding strand is the DNA strand that encodes...to the mRNA transcript produced. The antisense strand is referred to as the template strand or noncoding...noncoding strand, as this is the strand that is transcribed by the RNA polymerase. Figure 1: Simplified promoter...initiation of transcription, whereas transcription factors promote the initiation of transcription. The binding...template for the translation of a protein. RNA polymerase III — transcribes genes encoding transfer RNAs (tRNA... eukaryotic promoters, including transcription and the transcription complex. Educational...to DNA. RNA polymerase transcribes DNA to mRNA, which is ultimately translated into a functional protein...

... centrifuged to concentrate. Crude or concentrated virus can then be used to transduce the cells of interest...cis Central polypurine tract; recognition site for proviral DNA synthesis. Increases transduction efficiency...crossover event between the transfer plasmid and genes provided in trans . Transduction The process of artificially...need three plasmids (Figure 2): Transfer plasmid — contains transgene, sgRNA, or shRNA of interest flanked...Production Cloning Cloning your transgene, gRNA, or shRNA of interest into the transfer plasmid can be done with...recombination-deficient bacteria strains, such as NEB Stable cells. These strains reduce the frequency of homologous...described above (envelope, packaging, and transfer) are co-transfected into the HEK293T packaging cell line...

... culminated in the central dogma of molecular biology, that proteins are translated from RNA, which is...is transcribed from DNA. The Genetic Code The genetic code is a set of rules for translating the information..., double-stranded helix. To create the double helix, the nucleotides on the opposing strands of DNA form...proteins can be translated, the DNA is converted to RNA in a process called transcription. RNA uses the ...proteins are transcribed and processed into mature messenger RNA (mRNA), which is then translated into proteins...reference to any extrachromosomal heritable determinant. Plasmids are fragments of double-stranded DNA that typically...Promoter Region Drives transcription of the insert. The promoter recruits transcriptional machinery and can...

... interfere with transcription elongation. CRISPR Activation : Target the transcription start site. CRISPRa...fused to transcriptional activators that need to be within a given range of the transcription start site... perform by chemical transfection or electroporation. Expression can be transient, or you can generate...efficiently transfect. However, note that this method is most useful for cell lines that you can transfect at ...into cells. These methods are more transient than plasmid transfection or viral vectors, reducing the risk...your target cells are not transcribing DNA to mRNA, nor do they need to translate mRNA to protein. Instead...is helpful in cells that are difficult to transfect, transduce, or may not express common promoters. Browse...

...synthetic two-part transcription factors, where Cry2 and CIB1 are fused to a transcriptional activation domain...mutations to alter the absorption spectrum or adding trafficking signals to localize opsins to the cell membrane...has higher conductance at physiological pH and traffics well to the membrane. Excitatory (Depolarizing..., C1V1(t/t), ChrimsonR Red-shifted peak action spectra Examples: VChR1, C1V1(t/t), Chrimson, ChrimsonR...eNpHR, eNpHR2.0, eNpHR3.0 Red-shifted peak action spectra Example: Jaws Archaerhodopsins Archaerhodopsin-...Variants Opsin Type Variant Description Peak Response Spectra (nm) Channelrhodopsins: cation channels from Chlamydomonas...pump from Haloarcula (Halobacterium) salinarum (strain Shark), used to silence neuronal activity 632 Halo...

... neuronal signaling by increasing intracellular cAMP concentrations. There is currently one Gs-DREADD,...populations in Japan, making it an attractive option for translational studies, however, it has off-target...against hM4Di, and is especially attractive for use in translational studies because it is FDA and EMA...cell surface receptors, GPCRs are seven-pass transmembrane proteins that bind a specific ligand. Ligand...initiating signaling pathways in inflammation and neurotransmission. This, and the fact that GPCR ligands can ...important for dosing in animal studies, made them attractive targets for development into chemogenetic tools...stimulating phospholipase C, which releases intracellular calcium stores. There are currently three Gq...