分子遗传学理论与技术基础-Chapter-III-technology-basis第二部分.ppt

1、Chapter technology basis of molecular genetics Summary about the technology for gene research . isolation and purify of nuceic acid 1.Types of nucleic acid in biont:karyotas: chromosome DNA mitochondria DNA RNA moneras: chromosome DNA plasmid DNA RNA virus: DNA or RNA (that is enveloped by protein s

2、hell ) 2.Principle of isolation and purify to keep nucleic acid molecular structure complete to avoid nucleic acid sample polluted by protein or other nucleic acid to decrease the capacity of enzyme inhibitor in sample 3.To isolate and purify animal chromosome DNA(鄂 p271) tissue sources of genome DN

3、A:a: fresh tissue or operating sample p274b: blood sample P274c: cultured cells P271 basic reagents and their function: 鄂 p271-273Antithrombotics (抗凝剂 ) 2%EDTA EDTA 2g(乙二胺四乙酸 ) p274(鄂 p274) NaCl 0.85gadd H2O to 100 ml * ACD solution citric acid 0.48g (柠檬酸 )sodium citrate 1.32g(柠檬酸钠)glucose 1.47gadd

4、H2O to 100 mlVolume ration: blood:ACD=6:1n Buffer PBS （磷酸盐缓冲液） 鄂 P488TBS （ Tris-硼酸缓冲液） 鄂 P487n Cytolysis solution (细胞裂解液 ) 鄂 P27210mM Tris-Cl (PH7.4):【 this solution is regulated to PH7.4 by HCL solution (鄂 486表 )】10mM EDTA： 【 that is the inhibitor for DNase】150mM NaCl0.4%SDS(十二烷基硫酸钠 )【 to destroy b

5、iomembrane and be added finally】Quantitative volume to 1000ml by 10mM Tris-Cl finallyn Protase K : to degrade protein into oligopeptids.the work solution is 20mg/ml,storage at -20 .n Phenol : protein denaturant of biochemical pure can be used directly. （鄂 P485 卢 P51） chemical pure have to be redisti

6、lled.n Chloroform(氯仿 )： it cooperate with phenol to speed separate the water phase from organic phase. n NH4Ac: DNA sedimentation reagent.n Ethanol: to make DNA molecular dehydrate and separate out .n Ether(乙醚 )： to remove phenol and chloroform from DNA sample .n TE solution: it is DNA storage solut

7、ion 鄂 P486 10mM Tris-Cl1mM EDTA technology steps for isolating genome DNA from human blood sample:(need large volume DNA ) 鄂 P274 to get leucocyte cells from blood sample:fresh blood sample: ACD solution=6:1,mixed, 1300g centrifuge for 15min ,draw away the up layer of serum, then take up the yellown

8、ess cell layer of sediment into a new tube, avoid the red cell mixed (may repeat again) to destroy cell membrane, releasing genome DNA:-suspend leucocyte cells in cytolysis solution 15ml for 1h,and waterbathing at 37 to degrade protein of chromatin and liberating DNA molecules :-add 20mg/ml of prota

9、se K to 100ug/ml terminal concentration ,mixed to homogenous ,keep in vibrator(振荡仪 ) at 50 for 3h to remove protein and extract DNA into water phase:-cold to RT ,add equal volume of solution (phenol / chloroform =1:1),vibrate softly to make water phase and organic phase mixed, centrifuge 5000g for 15min, then extract solution is separated into 3 phases* in tube, to draw out the water phase carefully into a new tube with big bore pipette tube(0.3cm).repeat again to purify DNA.