Introducing a New and Straightforward Approach for DNA Purification from a Gel

Document Type : Research paper

Authors

1 Department of Agronomy and Plant Breeding Sciences, College of Aburaihan, University of Tehran

2 Department of Agronomy and Plant Breeding Sciences, University of Kurdistan- Faculty of Agriculture, Sanandaj, Iran

Abstract

In most procedures that involve gene cloning, after the amplification of a target gene by PCR or by Real-time PCR, the purification of the trapped
gene on agarose gel is a crucial stage. There are various methods for extracting genes from agarose gel by removing other contaminants. We isolated the amplified PqHMGR gene (derived from Ginseng (Panax quinquefolius)) from agarose gel by a quasi-electrophoresis device (similar to electro-elution technique). Moreover, the efficiency of this new approach was compared with that of the commercial kit ‘Silica Bead DNA Gel Extraction’ (Thermo Scientific American Company). Ligation to the PTG-19 plasmid and cloning in E. coli bacteria were also done. The results showed successful isolations of targeted DNA, along with a high efficiency in producing recombinant DNA and in concluding a successful cloning procedure through this new device. The invented method provided a better purification ability than the commercial kit, but because of using the TAE 1X buffer as the purified gene storage solution, the plasmid and bacterial transformation rates were slower than the commercial kit method. It was found that using the new method for the purification of nucleotide sequences by electrophoresis and electrophoresis buffer is feasible, and that these purified fragments can be applied in cloning and sequencing. Using the TAE 1X buffer instead of distilled water did not cause problems in gene binding to PTG-19 plasmid. It also allowed a successful transformation of E. coli bacteria by the modified plasmid. Nonetheless, using TAE 1X buffer reduced the modification rate of the PTG-19 plasmid and decreased the rate of E. coli transformation by the modified plasmid.
Abbreviations:
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), Complementary DNA (cDNA), Diethyl Pyrocarbonate (DEPC), Escherichia coli (E. coli), Ethylenediaminetetraacetic acid (EDTA), Isopropyl ß-D-1-thiogalactopyranoside (IPTG), LB (Luria Broth), Optical density 260 (OD260), Optical density 280 (OD280), Panax quinquefolius HMGR (PqHMGR), Polymerase chain reaction (PCR), Reverse transcription polymerase chain reaction (RT-PCR), Tris/Borate/EDTA (TBE), Tris-acetate-EDTA. 1X (TAE).

Keywords

References

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International Journal of Horticultural Science and Technology
Volume 9, Issue 4
October 2022
Pages 477-486

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History

  • Receive Date: 31 July 2021
  • Revise Date: 28 October 2021
  • Accept Date: 22 November 2021

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