摘要
取活鲤鱼肝胰脏,经冲洗后,立即投入液氮中.然后制成匀浆.先在4℃下600g离心,保留上层液.再900g离心,保留沉淀物并用不同溶液重复悬浮洗涤离心,可得到比较纯的线粒体.将上述线粒体在含1%SDS的Saline-Na_2EDTA溶液中悬浮,于37℃反应15min.然后在室温用混合溶剂萃取,先后用乙醇洗涤700g、1200g离心,待用电泳检查RNA除净后.加少量蛋白酶-K除去其余蛋白质,最终获得比较纯的DNA.用电镜检查DNA的完整性及用紫外分光光度计测定其浓度.
The liver-pancreas was rapidly removed from live carp and washed before being transferred into liquid nitrogen. The tissue homogenate was centrifuged at 4 C and 600g, and the supernatant was centrifuged once more at 900g. The pellet obtained was resuspended several times including centrifuging to obtain purified mitochondria.The above mitochondria pellet was then resuspended with saline-Na2EDTA, and incubated gently in 1% SDS for 15 min at 37 C. After chilling to room temperature, the suspension was then purified with phenol/chloroform exeraction and ethanol washing and centrifuging. while the pellet obtained was resusdended in 0. 1 X SSC buffer and suitable amount of RNase-A was added to remove RNA completely as judged with electrophoresis. Finally Proteinase- K was used to remove remaining protein in order to obtain purified mt DNA. The integrity of mt DNA was checked under electronmicroscopy and its concentration determined with UV spectrophotometry.
出处
《环境化学》
CAS
CSCD
北大核心
1994年第3期283-285,共3页
Environmental Chemistry
基金
国家自然科学基金资助项目