从大体积的PAGE凝胶中纯化长链寡核苷酸的新方法

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)%。

A New Method for the Recovery of Long Oligonucleotide from Bulky Polyacrylamide Gels

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)%.