鲤鱼肝胰脏线粒体DNA的分离,纯化方法

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游泳重复悬浮洗涤离心，可得互比较纯的线粒体。将上述粒体在含１％ＳＤＳ的Ｓａｌｉｎｅ－Ｎａ２ＥＤＴＡ溶液中悬浮，于３７℃反应１５ｍｉｎ，然后在室温用混合溶剂萃到，先后用乙醇洗涤７００ｇ，１２００ｇ离心，待用电泳检查ＲＮＡ除净后，加少量蛋白醇－Ｋ除去其余蛋白质，最终获得比较的Ｄ

METHOD OF ISOLATION OF MITOCHONDRIA DNA FROM CARP LIVER -PANCREAS

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.