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THE OPTIMIZATION OF CLONING OF LONG-SIZED DNA FRAGMENTS IN p-GEM VECTOR, CONTAINING TERMINAL INVERTED REPEATS OF LINEAR MITOCHONDRIAL PLASMIDS FROM ZEA MAYS
Original title
ОПТИМИЗАЦИЯ КЛОНИРОВАНИЯ ДЛИННОРАЗМЕРНЫХ ФРАГМЕНТОВ ДНК В КОНСТРУКЦИИ p-GEM, СОДЕРЖАЩЕЙ КОНЦЕВЫЕ ИНВЕРТИРОВАННЫЕ ПОВТОРЫ ЛИНЕЙНЫХ ПЛАЗМИД ИЗ МИТОХОНДРИЙ ZEA MAYS
Authors
Panfilov A.V.1, Konstantinov Yu.M.1,2, Koulintchenko M.V.2
Contact information
1Irkutsk State University, Irkutsk, Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.
2Siberian Institute of Plant Physiology and Biochemistry SB RAS, Irkutsk, Russia, e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.
Pages
84-86
DOI
10.31255/978-5-94797-318-1-84-86
Abstract
Optimization of cloning conditions (reaction space, thermodynamic and kinetic factors) of DNA molecules with long size is an important practical and experimental task in modern molecular biology, genetic research and structural biochemistry. Cloning allows manipulate DNA sequences, which facilitates matrix synthesis of given molecular structures, as double-strand and circular DNA molecules. The aim of this work is to optimize conditions of cloning of DNA molecules with 7-10 kb length in genetic structures pGEM-IRSI-IR2.3 and pGEM-IR2.3, containing terminal inverted repeats of the linear mitochondrial plasmid of maize (Zea mays), SI (6.4 kb.) and S3 (2.3 kb). The sequence of a DNA fragment from the mitochondrial genome of Brassica napus was amplified in PCR using oligonucleotides containing recognition sites for restriction endonucleases and different amplification enzymes. The obtained PCR products were purified using spin columns contaning silicone carrier or by extraction with water-organic phase of phenol-chloroform-isopropanol solution. To optimize the nucleic acids hydrolysis conditions by restriction endonucleases, the reaction mixture volume and reaction time were tested.