线粒体DNA的提取方法.

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1、 线粒体线粒体DNA的提取的提取方法 Protocol for Extracting Mitochondria DNA protocol 从植物组织提取高纯度线粒体DNA 实验目的1、4000rpm 离心可以沉淀分离出叶绿体、细胞核和组织碎片，12,000 rpm离心才可沉淀分离线粒体。实验原理3、SDS可以从植物各样组织类型中释放和结合的细胞核酸。2、用蔗糖浓度梯度纯化线粒体，不同浓度蔗糖的缓冲液因比重不同而分层。研钵和研杵；冷冻离心机；过滤装置（漏斗，玻棒）pH计；移液器；水浴箱；50mL离心管；2；1.5 mL离心管。Apparatus:Apparatus:仪器设备仪器设备：mortar

2、(cold);Centrifuged;filter;pH meter;water bath boiler;50mL centrifugal tube;eppendorf tube.试剂：试剂：试剂：试剂：Reagent and solutionReagent and solutionReagent and solutionReagent and solution A buffer:10 mmol/L Tris-HCL，pH 7.4；500 mmol/L；20 mmol/LEDTA；0.2%BSA；0.2%-mercaptoethanolB buffer:10 mmol/L Tris-HCL，p

3、H 7.4；600 mmol/L sucrose;20 mmol/L EDTALysis buffer:50 mmol/L Tris-HCL，pH8.0；20 mmol/L EDTA试剂：试剂：试剂：试剂：Reagent and solutionReagent and solutionReagent and solutionReagent and solution 缓缓冲冲液液A：10 mmol/L T ris-HCL，pH 7.4；500 mmol/L 蔗蔗糖糖；20 mmol/LEDTA；0.2%BSA；0.2%-巯基乙醇巯基乙醇缓缓冲冲液液B：10 mmol/L T ris-HCL，pH

4、 7.4；600 mmol/L 蔗蔗糖糖；20 mmol/L EDTA 裂裂解解缓缓冲冲液液：50 mmol/L T ris-HCL，pH8.0；20 mmol/L EDTA操作步骤操作步骤 线粒体的提取及纯化线粒体的提取及纯化Mitochondria isolationProtocol1.1.将一定鲜重（将一定鲜重（将一定鲜重（将一定鲜重（FW FW）的黄化苗剪成的黄化苗剪成的黄化苗剪成的黄化苗剪成23 cm 23 cm 的小段，倒的小段，倒的小段，倒的小段，倒入入入入5 ml/g FW5 ml/g FW的缓冲液的缓冲液的缓冲液的缓冲液A A，然后在冰水浴（以下未加说，然后在冰水浴（以下未加

5、说，然后在冰水浴（以下未加说，然后在冰水浴（以下未加说明，操作温度均为明，操作温度均为明，操作温度均为明，操作温度均为0 40 4）中进行研磨。研磨液用）中进行研磨。研磨液用）中进行研磨。研磨液用）中进行研磨。研磨液用8 8 层层层层纱布过滤，滤液经纱布过滤，滤液经纱布过滤，滤液经纱布过滤，滤液经4 000rpm 4 000rpm 离心离心离心离心15 min15 min。1.Harvest 1.Harvest 10g 10g dark-grown dark-grown etiolated etiolated seedlings,seedlings,cool cool them to 0the

6、m to 0.Conduct the subsequent operations on ice.Conduct the subsequent operations on ice.Homogenize Homogenize the the seedingsseedings in in the the mortar mortar with with 50ml 50ml of of A A buffer.buffer.Filter Filter the the homogenate homogenate through through(8-layer(8-layer muslin muslin cl

7、oth)cloth)and and centrifuge centrifuge the the extract extract at at 4000rpm,4000rpm,for for 15 15 min.min.2.2.取取取取中中中中的的的的上上上上清清清清，再再再再经经经经12 12 000rpm000rpm离离离离心心心心20 20 minmin。弃弃弃弃上上上上清清清清，往往往往此此此此沉沉沉沉淀淀淀淀中中中中加加加加入入入入0.0.1 1 mL/gmL/g FW FW 的的的的缓缓缓缓冲冲冲冲液液液液A A，用用用用毛毛毛毛笔笔笔笔轻轻轻轻轻轻轻轻悬浮，悬浮液再经悬浮，悬浮液再经

8、悬浮，悬浮液再经悬浮，悬浮液再经 4 000rpm 4 000rpm 离心离心离心离心15 min15 min。2.Discard 2.Discard the the nuclei nuclei pellet,pellet,centrifuge centrifuge the the supernatant supernatant at at 12,000rpm,12,000rpm,for for 20 20 min.min.Re-suspend Re-suspend the the pellet pellet gently gently by by using using a a soft s

9、oft paintbrush,paintbrush,centrifuge centrifuge at at 12,000rpm,12,000rpm,for for 20 20 min.min.Re-suspend Re-suspend the the organelle organelle pellet pellet with with a a soft soft paintbruspaintbrus in in minimal minimal volume volume of of A A buffer buffer(0.(0.1 1 mL/gmL/g FW).Centrifuge at 4

10、000rpm,for 15 min.FW).Centrifuge at 4000rpm,for 15 min.3.取中的上清，加入DN ase I 及M gCL 2，使两者的最终浓度分别为10 g/g FW 和10 mmol/L，在4下反应1 h，再加入EDTA 至100 mmol/L，以终止DN ase I 的反应。Discard the residue,DNase-1 and MgCL2 were added to the plant material to make a final concentration 10 g/g FW of material and 10 mmol/L.The

11、 DNase-1 treatment done at 4 for 1h(or at at 37 for 30minutes.Adding EDTA-Na2 to final concentration of 100 mmol/L stopped the raction.B-buffer were added slowly to the tube bottom making a final concentration 0.2mL/g FW of material.Collect the organelle pellets by centrifugation,12,000rpm,for 20 mi

12、n.4.利用蔗糖衬垫法纯化线粒体，即在中的离心管底部缓缓加入0.2mL/g FW 的缓冲液B(10 mmol/L T ris-HCL，pH 7.4；600 mmol/L 蔗糖；20 mmol/L EDTA)，再经12 000rpm离心20 min。Re-suspend Re-suspend the the organelle organelle pellet pellet with with a a soft soft paintbruspaintbrus in in minimal minimal volume volume of of A A buffer buffer(0.(0.1 1

13、mL/gmL/g FW),FW),B B-buffer were added slowly to the tube bottom making a final concentration 0.2mL/g FW of material again.Collect the organelle pellets by centrifugation,12,000rpm,for 20 min.Keep the pellet at-20 or lyse it at once Keep the pellet at-20 or lyse it at once 5.弃弃上上清清，沉沉淀淀中中加加入入0.1 mL/

14、g FW 的的缓缓冲冲液液A，离离心心管管底底部部再再缓缓缓缓加加入入0.2 mL/g FW 的的缓缓冲冲液液B，12 000rpm离心离心20 min，所得沉淀即为纯化的线粒体所得沉淀即为纯化的线粒体 LysisLysis and Purification and Purification 线粒体线粒体线粒体线粒体DNA DNA 的提取及纯化的提取及纯化的提取及纯化的提取及纯化 1.1.在纯化后的线粒体沉淀中加入在纯化后的线粒体沉淀中加入在纯化后的线粒体沉淀中加入在纯化后的线粒体沉淀中加入0.1 Ml/g FW 0.1 Ml/g FW 裂解液裂解液裂解液裂解液(50(50 mmolmmol/

15、L T/L T risris-HCL-HCL，pH8.0pH8.0；20 20 mmolmmol/L EDTA)/L EDTA)，轻轻混匀沉淀，再加入蛋白酶轻轻混匀沉淀，再加入蛋白酶轻轻混匀沉淀，再加入蛋白酶轻轻混匀沉淀，再加入蛋白酶K(10mg/mL)K(10mg/mL)和和和和10%10%的的的的SDS(SDS(用裂解液配制用裂解液配制用裂解液配制用裂解液配制)，使两者的最终浓度分别为，使两者的最终浓度分别为，使两者的最终浓度分别为，使两者的最终浓度分别为10 10 g/g FW g/g FW 和和和和2%.2%.1.1.ResuspendResuspend the the pellet

16、pellet in in in in lysislysis-buffer-buffer(0.(0.1 1 Ml/g Ml/g FW FW).).Protein Protein K K and and 10%10%SDS SDS added added to to the the plant plant material material to to make a final concentration 10 g/g FW and 2%.make a final concentration 10 g/g FW and 2%.2.The material were lysed in 2%SDS,c

17、onstantly shaking the mixture.If the tissue coalesces,warm the tubes in a 60water bath for 40 min(or in a 37water bath for 4h)until the organelles are fully lysed.Shake the mixture intensively for 10min with an equal volume of equilibrated phenol.Centrifuge for 10min at 12,000 rpm.Remove the upper a

18、queous phase.The RNase treatment was done at 37 for 30minutes.Then add phenolchloroformisoamyl alcohol（25241）.Repeat the procedure.Add chloroformisoamyl alcohol（241）.Repeat the procedure.Precipitate the DNA with 1/10 vol of 3M NaAC and 2 vol of isopropanol at-20 for 2-3h or overnight.2.2.线线粒粒体体在在373

19、7下下裂裂解解4 4 h h后后(或或6060下下裂裂解解40min)40min)，往往裂裂解解液液中中加加入入等等体体积积的的酚酚-氯氯仿仿异异戊戊醇醇，轻轻轻轻混混匀匀，静静置置5 5 m m inin。在在12 12 000rpm 000rpm 44下下离离心心10 10 minmin。吸吸出出上上清清，再再用用酚酚-氯氯仿仿/异异戊戊醇醇抽抽提提，吸吸取取上上清清，加加入入?RNaseRNase,在在3737下下保保温温30m 30m inin后后,加加入入酚酚-氯氯仿仿/异异戊戊醇醇抽抽提提，吸吸出出上上清清，再再用用氯氯仿仿/异异戊戊醇醇抽抽提提。吸吸出出上上清清,加加入入1/10

20、 1/10 体体积积的的乙乙酸钠和两倍体积的异丙醇，置酸钠和两倍体积的异丙醇，置-20 2 h-20 2 h 或过夜。或过夜。3.3.将将沉沉淀淀有有mtDNAmtDNA 的的离离心心管管经经12 12 000rpm000rpm、4 4 离离心心16 16 m m inin，回回收收DNA DNA 沉沉淀淀，再再用用70%70%的的乙乙醇醇漂漂洗洗沉沉淀淀1 1 2 2次次，沉沉淀淀以以室室温温凉凉干干，溶溶于于适适当当体体积积的的TE TE 缓缓冲冲液液或或无无菌去离子水中待用。菌去离子水中待用。3.Centrifuge 3.Centrifuge at at 12,000rpm12,000rpm，4 4 for for 16 16 min.min.Wash Wash the the DNA DNA pellet pellet with with 70%70%ethanol,ethanol,dry dry at at room room temperature and re-dissolve in TE-buffer.temperature and re-dissolve in TE-buffer.