Abstract
In this article, we present several protocols that describe the steps from cloning and obtaining a large amount of pure plasmid DNA to generation of lentiviruses based on these constructs. The protocols have been worked out on human cell culture HEK293T but can be adapted for other cell cultures. This protocol was designed to be simple to execute and cheap since it requires only materials and consumables widely available in molecular laboratories, such as salts, alcohols, etc., and no complicated laboratory equipment. These protocols are highly effective and can be performed in any standard molecular biology laboratory.
Highlights
Transfection is a procedure that introduces DNA and RNA into eukaryotic cells to produce genetically modified cells
Transfection can be used in gene therapy, for production of recombinant proteins for therapeutic purposes, small interference
Our method can significantly reduce the cost of DNA isolation, since it provides a high yield of extracted plasmid without an implementation of commercial kits
Summary
Transfection is a procedure that introduces DNA and RNA into eukaryotic cells to produce genetically modified cells. Transfection is a powerful analytical tool for studying gene function and their regulation by enhancing or inhibiting specific gene expression in cells. It is important for studying proteins or producing recombinant proteins in mammalian cells [1]. Our method can significantly reduce the cost of DNA isolation, since it provides a high yield of extracted plasmid without an implementation of commercial kits. For this method, we used the E. coli bacterial strain STBL3 (ThermoFisher Scientific, Walthon, MA, USA). Transfection using PEI has several advantages, like its cheapness and suitability for various cell cultures [4]
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