How to get template for in vitro transcription?
Best insight from top research papers
To obtain a template for in vitro transcription, several methods can be employed based on the desired RNA transcript characteristics. One approach involves using synthetic DNA templates containing target mRNA sequences flanked by bacteriophage promoters, which are then amplified by PCR for subsequent transcription . Another method utilizes ligase-independent preparation of long synthetic DNA templates through Recursive PCR, allowing for easy incorporation of long insertions, mutations, or deletions before transcription . Additionally, in vitro transcription-translation systems can utilize bacterial genomes as templates to mimic intracellular RNA and protein profiles, showcasing the potential of using genomes for in vitro transcription . Lastly, for studying transcription on chromatin templates, a protocol involving nucleosome assembly on ribosomal RNA minigenes is described, providing a tool to analyze transcriptional dynamics on chromatin substrates .
Answers from top 5 papers
More filters
| Papers (5) | Insight |
|---|---|
19 Citations | Linearized plasmid DNA with a T7 promoter upstream of the gene to be transcribed is used as the template for in vitro transcription. |
2 Citations | To obtain a template for in vitro transcription, assemble nucleosomes on ribosomal RNA minigenes using salt gradient dialysis and sucrose gradient centrifugation, as outlined in the protocol. |
13 Citations | The template for in vitro transcription can be obtained by using bacterial genome as a template, as demonstrated in the study on in vitro transcription-translation using bacterial genome. |
10 Citations | Long templates for in vitro transcription can be prepared using Recursive PCR, where complementary DNA oligonucleotides are annealed, extended, and amplified in a single PCR reaction. |
| DNA templates for in vitro transcription can be obtained by amplifying synthetic DNA containing target mRNA sequences flanked by bacteriophage promoters through a single round of PCR. |
Related Questions
Is RNA stable in vitro?5 answersRNA stability in vitro varies depending on the specific conditions and factors involved. Studies have shown that RNA can maintain stability under certain circumstances. The stability of RNA duplexes in crowded environments has been investigated, with parameters determined for predicting stability accurately. Research on stress granules has highlighted the role of RNA helicases in regulating RNA-RNA interactions, impacting RNA condensate formation and stability. In contrast, microRNA has been found to be relatively stable compared to mRNA under defined ribonuclease treatment conditions, suggesting differential stabilities among RNA classes. Additionally, RNA folding stability within live mammalian cells has been compared to in vitro conditions, showing variable stability influenced by cellular effects. Overall, RNA stability in vitro is influenced by a range of factors, highlighting the complexity of RNA behavior in different environments.
What is Template-Based Computational methods in proteins?4 answersTemplate-based computational methods in proteins involve using 3D signatures called structural templates to identify and analyze protein functional sites such as ligand binding cavities, metal coordination motifs, or catalytic sites. These templates are generated using algorithms that query protein structures for conserved 3D motifs. By using more than one template per structure, flexible sites can be represented, allowing for a better understanding of catalysis through snapshots captured in enzyme structures. These methods are important for functional annotation from protein structure, especially with the advancements in protein structure prediction. Template-based modeling is a popular approach for predicting protein tertiary structure, where deep learning techniques are used to accurately align query sequences with templates and select appropriate templates for distant homology proteins. These methods have shown promising results in improving the accuracy of structure prediction, particularly for proteins with only distant homologs available.
What are some examples of in vitro studies?5 answersIn vitro studies have been used in various fields of research. For example, stem cell models derived from human pluripotent stem cells have been used to study chronic long-term toxicity and the onset and progression of diseases such as Alzheimer's disease and epilepsy. In the pharmaceutical industry, in vitro models have been utilized for preclinical screening of drug metabolism, drug-drug interactions, and the effect of environmental agents, ethnic background, gender, and genetics on drug metabolism. In the field of ACL research, in vitro models have been used to study cellular behaviors and response to environmental cues, providing insights for the development of new treatment strategies. In ethnopharmacological studies, in vitro tests have been used to investigate the biological activity of plant extracts and their potential therapeutic uses. In the field of dermatology, in vitro studies have been conducted using cell culture models to study paracrine or contact cell-cell effects, as well as the maturation and differentiation of melanocytes.
What is the procedure for producing cotton fiber in vitro?5 answersThe procedure for producing cotton fiber in vitro involves several steps. First, the cotton fibers are obtained by culturing cotton ovules in a defined medium containing auxin and gibberellic acid. This allows the fibers to continue developing and producing trichomes in a controlled environment. Next, various substances or solutions are used to enhance the growth and properties of the cotton fibers. For example, alkyl imidazole is dissolved in an aqueous solution, and cotton is immersed in this solution along with a strong alkaline solution. Another method involves impregnating cotton fiber into a mixed solution of titanium salt, organic acid, and ethanol. Additionally, a culture medium can be used to increase the length of cotton fibers by adding a second component to a BT culture medium. Finally, a preparation method involves drawing bamboo fiber and silk protein fiber, immersing them in a NaOH solution, and then drying and spinning them to obtain hygroscopic and sweat releasing down cotton fiber.
What are the requirements for establishing an in vitro meristem culture?5 answersIn order to establish an in vitro meristem culture, several requirements need to be considered. First, the specimens should be sterilized using a suitable concentration of sodium hypochlorite (NaOCl) for a specific duration. The choice of plant growth regulators is crucial for the successful micropropagation of plants. Different combinations of cytokinins (such as BAP and kinetin) and auxins (such as NAA) can be used to promote shoot proliferation and root induction. The choice of medium is also important, with MS (Murashige and Skoog) basal medium being commonly used. Additionally, the use of liquid or semi-solid/liquid double layer media can affect the efficiency of shoot induction and proliferation. Finally, the establishment of the regenerated plantlets in soil containing organic fertilizers is necessary for their successful growth and development.
What are some examples of in vitro experiments?5 answersIn vitro experiments have been widely used for more than a century to elucidate molecular mechanisms in cells and pathogen-host interactions, as well as for drug screening. A variety of in vitro methods have been developed, including inert tissue-mimicking phantoms, cell-based methods, blood clots, collagen gels, and 3D biological scaffolds. In the field of bone biomechanics, in vitro experiments have been used to preliminarily identify failure scenarios, improve model identification, and provide quantitative validation. In ethnopharmacological studies, in vitro tests are used to investigate the activity of substances used in traditional medicine, where limited supplies of material are available. In the field of ACL research, in vitro models are used to study cellular behaviors under controlled conditions before translating to preclinical and clinical research.