摘要
为解决烤后烟叶基因组DNA提取稳定性差的问题,以烤后烟叶为材料,对CTAB法提取基因组DNA进一步改良优化:对CTAB沉淀缓冲液使用量、裂解时间、Tris平衡酚的处理次数和RNase处理时间4个影响因素进行单因素优化.实验结果表明:CTAB沉淀缓冲液使用DNA溶液体积的1.5倍、裂解时间40 min、Tris平衡酚抽提1次和RNase(10 mg/mL,1μL)处理10 min,能得到主带清晰的烤后烟叶基因组DNA.该方法提取得到的烤后烟叶基因组DNA OD260/OD280为1.7～1.9,OD260/OD230>2.0,35 ng左右基因组DNA即能得到清晰的RAPD和SCAR图谱,适用于以PCR为基础的分子生物学实验.
The experimental method which improves the CTAB for extracting the genomic DNA in flue- cured tobacco was presented in order to stabilize the extraction of the genomic DNA. Each of the four influ- encing factors in the CATB, namely, the usage amount of precipitate buffer solution, pyrolysis time, num- ber of processing the Tris phenol and the processing time for RNase were optimized respectively. The re- suits showed that after using CATB precipitate buffer solution with the volume 1.5 times larger than that of the DNA solution with pyrolysis time for 40 min, one-time extraction of the Tris phenol and 10 min pro- cessing the Rnase（10 mg/mL,1 μL） , a clear main band of the genomic DNA in the flue-cured tobacco could be obtained. The extracted genomic DNA OD260/OD280 was between 1.7 and 1.9. OD260/OD230 was greater than 2.0. In this case, with 35 ng genomic DNA, clear RAPD and SCAR maps could be obtained. The extracted genomic DNA can be applied to the molecular biological experiments.
出处
《郑州轻工业学院学报（自然科学版）》
CAS
2013年第2期59-63,共5页
Journal of Zhengzhou University of Light Industry:Natural Science
