Mohd-Hairul A R, Sade A B, Yiap B C, Raha A R
Department of Cell and Molecular Biology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
Genet Mol Res. 2011 Nov 8;10(4):2757-64. doi: 10.4238/2011.November.8.1.
DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.
使用TriOmic™提取试剂盒对马来西亚现有的32个药用植物样本进行DNA提取。取0.1克花朵或幼叶，用液氮研磨，在65°C的RY1（plus）缓冲液中裂解，随后进行核糖核酸酶处理。然后，向样本中加入RY2缓冲液，通过涡旋充分混合，再通过离心去除细胞碎片。将上清液转移到新的微量离心管中，向每个转移的上清液中加入0.1体积的RY3缓冲液。将混合物应用于旋转柱，随后进行离心步骤以去除缓冲液和其他残留物。通过向旋转柱中加入70%乙醇进行两次洗涤步骤。通过向每个旋转柱的膜上加入50 μL TE缓冲液，随后在室温下进行离心步骤，回收样本的基因组DNA。在提取过程中加入氯仿：异戊醇（24:1）步骤，对TriOmic™提取程序进行了改进。当在TriOmic™提取程序中应用氯仿：异戊醇（24:1）步骤时，从32个样本中的大多数提取的基因组DNA的总产量有所增加。这项初步研究对于马来西亚现有药用植物的分子研究非常重要，因为与手动提取相比，DNA提取可以在更短的时间内（1小时内）完成，手动提取需要使用苯酚、氯仿和乙醇沉淀，需要1至2天才能完成。
