Method for purifying plasmid DNA on calcium phosphate compound

#1. Field of the Invention

#The present invention relates to a method for isolating plasmid deoxyribonucleic acid (DNA) from nucleic acids which is one of the most basic operations in the field of genetic engineering.

#2. Description of the Related Art

#PBulescriptSK(+) (trademark of Stratagene) having an ampicillin-resistant gene was transformed to a JM 109 competent cell which was then cultured in a LB-agar medium containing 50 .mu.g/mL of ampicillin at 37.degree. C. for a whole day and night to obtain a single colony species. The colony species was then cultured by being shaken in an LB culture solution containing 50 .mu.g/mL of ampicillin at 37.degree. C. for a whole day and night to obtain a bacterium body. After centrifugal separation, the bacterium body was treated with an alkali-SDS method to prepare a mixed solution of plasmid DNA and RNA having a nucleic acid concentration of 200 .mu.g/mL (hereinafter, referred to as a "test solution").

#A tris-calcium salt buffer solution having a pH, calcium chloride having a concentration and a buffer solution of tris(hydroxymethyl)aminomethane-hydrochloride having a concentration shown in the following Table 1 was prepared.

#Thereafter, hydroxyapatite particles having an average particle diameter of about 20 microns and a specific surface area, determined in accordance with the BET method, of about 50 m.sup.2 /g, were suspended in 70 .mu.L of the tris-calcium salt buffer solution having the described pH, so that a suspension having a concentration of 10% (w/v) was obtained. Then, 10 .mu.L of the test solution was added to 0.2 mL of the obtained suspension and the suspension was shaken for five minutes at room temperature. Thereafter, a supernatant was recovered from the suspension, and then subjected to a gel electrophoretic process using 0.8% agarose gel. An analysis of the nucleic acid components was made with dying using ethidium bromide to obtain the results shown in the following Table