荸荠基因组DNA的提取及RAPD反应体系的建立(英文)

Extraction of genomic DNA and establishment of RAPD reaction system for water chestnut(Eleocharis tuberose Roem et Schult)

以荸荠叶状茎为试验材料,采用改良的SDS法提取其基因组DNA,并对其RAPD反应体系进行优化,建立了荸荠的RAPD-PCR优化反应体系和程序。结果表明,提取的基因组DNA纯度和完整性较好,OD260/OD280值在1.8～2.0之间,DNA无降解现象,完全可以满足RAPD-PCR扩增要求。建立了荸荠RAPD反应体系:总体积为25μl,各有关成分的最佳浓度分别为25mmol/L Mg2+,1.0U Taq DNA聚合酶,0.2mmol/L dNTPs,1μmol/μl引物,1.5ng/μl DNA模板。PCR反应程序为:94℃预变性3min;94℃变性1min,37℃退火30s,72℃延伸60s,40个循环;最后72℃延伸10min。

The young phylloclade of water chestnut was used as experimental material, and the improved SDS method was used to extract the genomic DNA of young phylloclade successfully. The results showed that the extracted genomic DNA was pure and integral, and the ratio of OD260/OD280 was from 1.8 to 2.0, and the degradation of extracted genomic DNA was not found. Therefore, the extracted genomic DNA was suitable for RAPD-PCR reaction. The conditions for RAPD-PCR were optimized, and the best RAPD reaction system was established as follows： total volume being 25μl, containing 25 mmol/L Mg^2＋, 1.0 U Taq DNA polymerase, 0.2 mmol/L dNTPs, 1 μmol/μl primer and 1.5 ng/μl template DNA. The PCR program was 3 min at 94℃ for predenaturalization, then 40 cycles of lmin at 94℃ for denaturation, 30s at 37℃ for annealing and 60s at 72℃ for extension, finally extension at 72℃ for 10min.