CAJ | 학술논문

通过比较从大体积的PAGE凝胶中回收寡核苷酸的不同方法,提出回收未银染的长链寡核苷酸的最佳方案:用12 cm × 1.5 cm的PAGE凝胶柱分离DNA,然后将凝胶切成长约0.5 cm的胶条,通过PCR反应挑选含有寡核苷酸的胶条.用2 mL的寡核苷酸洗脱缓冲液回收寡核苷酸,反复提取3次.用装填30 mg硅胶的C18 Sep-Pak反相层析柱进行纯化,最后经过离心冻干后即得到纯化的寡核苷酸,该方法的回收率可达(28.7±2.0)%.
통과비교종대체적적PAGE응효중회수과핵감산적불동방법,제출회수미은염적장련과핵감산적최가방안:용12 cm × 1.5 cm적PAGE응효주분리DNA,연후장응효절성장약0.5 cm적효조,통과PCR반응도선함유과핵감산적효조.용2 mL적과핵감산세탈완충액회수과핵감산,반복제취3차.용장전30 mg규효적C18 Sep-Pak반상층석주진행순화,최후경과리심동간후즉득도순화적과핵감산,해방법적회수솔가체(28.7±2.0)%.
By comparing different approaches for recovering small amount of long oligonucleotides from bulky polyacrylamide gels, a best method was suggested for purifying unstained long oligonucleotide as follows: Separate DNA in 5% polyacrylamide gels on 12 cm × 1.5 cm columns. Estimate the position of the oligonucleotide from the positions of tracking dyes. Cut the gel with a sharp, clean scalpel or razor blade. Every gel slice was about 0.5 cm in length and about 1 mL in volume. Transfer the gel slices to different 50 mL centrifuge tubes. Select polyacrylamide gel slices containing the oligonucleotide by PCR reaction. Extract DNA from the gel slices with 2 mL of oligonucleotide elution buffer by incubating the tubes at 37°C for 1 hour in a shaker incubator. Repeat to extract the oligonucleotide three times. Purify the recovered oligonucleotide with C18 Sep-Pak reversed-phase columns filled with 30 mg matrix. Finally, evaporate DNA solution to dryness in a centrifugal evaporator. The recovery rate of the oligonucleotide reached (28.7±2.0)%.