摘要
本文以中国角蟾属 4个种的蝌蚪酒精固定标本为材料 ,取尾部肌肉组织 0 1g,滤纸吸干组织块表面的酒精 ,在研钵中用剪刀剪碎 ,液氮研磨成粉 (必要时可加少量石英砂 ) ,转入 1 5ml离心管 ,加入 1 0ml提取缓冲液( 0 5 %SDS ,10mmol/LTris HCl,10 0mmol/LEDTA ,pH 8 0 ) ,37℃温浴h ,5 0 0 0r/min离心 10min ,取上清转入另一 1 5ml离心管 ,氯仿 /异戊醇 ( 2 4:1)抽提两次 ,上清液加 2倍体积预冷 ( - 2 0℃ )的无水乙醇沉淀DNA ,12 0 0 0r/min离心 3min ,无菌条件下风干后 ,5 0 μlTE溶解 ,4℃保存备用。以通用引物PCR扩增 12SrRNA和 16SrRNA基因部分片段并测序。本文不采用蛋白酶K提取酒精保存标本的DNA模板 ,全部流程只需 3个小时 ,可同时提取数十个乃至数百个标本 ,并且所提取的DNA模板适合短片段PCR扩增。
Alcohol-preserved tadpole samples of 4 species of Atympanophrys were studied. 0.1 gram tissue sample taking from tadpole tail muscle was cut into small particles and ground to fine powder with pestle in liquid nitrogen. The powder was allotted into Eppendorf tube and added extraction buffer (10 mmol/L Tris-HCl, 100 mmol/L EDTA, pH 8.0, 0.5% SDS). Then the tube was rocked gently and incubated at 37℃ for 1～2 hours. After incubation, the DNA was extracted with chloroform/isomylol (24/1) and centrifuged at 5000 rpm for 10 minutes. The top water phase was removed to a new tube and the extraction was repeated once again. The DNA in the top water phase was precipitated in -30℃ ice ethanol and pelleted by centrifugation at 12000 rpm for 3 minutes. The supernatant was poured off and the pellet was dried in air. The DNA was dissolved in 50μl TE buffer. The authors used those total DNA as template to successfully amplify 12SrRNA, 16SrRNA of the mitochondria DNA by PCR.
出处
《四川动物》
CSCD
2003年第2期66-68,共3页
Sichuan Journal of Zoology
基金
国家自然科学基金资助项目 ( 30 0 70 0 90 )
中国科学院西南基地创新工程资助。
