| 摘要 |
PURPOSE:To obtain an arbitrary DNA fragment having blunt terminal in high yield, by combining two short-chain oligomers with a DNA polymerase reaction. CONSTITUTION:For example, a DNA containing the desired double-stranded DNA fragment is cloned. 1pmol. of the obtained substrate DNA, 200pmol. each of short-chain DNA oligomers having chain A and chain B corresponding to a part of separated positions of a double-stranded or single-stranded DNA and 200nmol. of dNTPs are mixed with 10mul of a buffer solution (Composed of 100mM of Tris-HCl of pH 7.5, 500mM of NaCl and 100mM of MgCl2) and the mixture is diluted to 100mul. The product is thermally denaturated at 95 deg.C for 2-5min, annealed at 30 deg.C for 2min, added with 1 unit of Klenow enzyme and subjected to extension reaction at 30 deg.C for 3min. The above series of reaction is repeated 5 times or more to obtain a double-stranded DNA fragment. The DNA is integrated into a vector plasmid optionally after cutting the inner part with a restriction enzyme. |
