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The T7 promoter region allows large-scale in vitro T7 transcription to transcribe the DNA library into an mRNA library, which provides templates for the in vitro translation reaction later. The ribosomal binding site in the 5’-untranslated region (5’ UTR) is designed according to the in vitro translation system to be used.
NEB provides purification kits for both DNA and RNA. [23] [24] In May 2019, NEB released the Monarch Genomic DNA Purification Kit which is designed to minimize RNA contamination and allow high-yield purification of large DNA fragments. [23] NEB’s nucleic acid purification products have been used in various studies, including:
In vitro compartmentalization (IVC) is an emulsion-based technology that generates cell-like compartments in vitro. These compartments are designed such that each contains no more than one gene . When the gene is transcribed and/or translated , its products ( RNAs and/or proteins ) become 'trapped' with the encoding gene inside the compartment.
Run-off transcription can be used to quantitatively measure the effect of changing promoter regions on in vitro transcription levels, [1] [2] [4] Because of its in vitro nature, however, this assay cannot accurately predict cell-specific gene transcription rates, unlike in vivo assays such as nuclear run-on. [1] [2]
Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The in vitro protein synthesis environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability. [ 1 ]
A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. [1] RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter.
The polymerase is a monomeric protein with two distinct functional domains. Site-directed mutagenesis experiments support the proposition that this protein displays a structural and functional similarity to the Klenow fragment of the Escherichia coli Polymerase I enzyme; [3] it comprises a C-terminal polymerase domain and a spatially separated N-terminal domain with a 3'-5' exonuclease activity.
A nuclear run-on assay is conducted to identify the genes that are being transcribed at a certain time point. Approximately one million cell nuclei are isolated and incubated with labeled nucleotides, and genes in the process of being transcribed are detected by hybridization of extracted RNA to gene specific probes on a blot. [1]