实验二十五基因重组实验Ⅳ——重组体的筛选与鉴定

实验二十五基因重组实验w——重组体的筛选与鉴定［原理］限制性内切核酸酶酶切鉴定具体原理见实验二十一反应鉴定具体原理见实验十八PCR［试剂］、模板1DNA、引物

2、聚合酶3TaqDNA、（、、、）4dNTPs dATPdGTP dCTPdTTP、反应缓冲液510X PCR100mmol/LTris-HClpH

8.3,明胶500mmol/L KC1,15mmol/LMgC12,

0.1%、矿物油

6、超纯水

1、酶解反应液（）8EcoR I10X lmol/LpH

7.5Tris-HCl,

0.5mol/L NaCl,O.lmol/L MgCh、“血酶解反应液（）9din10X Imol/L pH

7.4Tris-HCl,Imol/LNaCl,

0.07mol/L MgCh电泳缓冲液称取克加入冰乙酸，

3.50XTAE242Tris,

57.1ml调节至加蒸储水至100ml

0.5mol/L EDTA,pH

8.0,1000mL、.凝胶加样缓冲液漠酚蓝、二甲苯青、

100.25%

0.25%FF40%蔗糖水溶液（）w/v、（）琼脂糖凝胶含（）琼111%

1.5%1XTAE100ml lg

1.5g脂糖［器材］、扩增仪1DNA、电泳仪、电泳槽

2、微量移液器

3、紫外投射仪

4、塑料离心管

50.5ml、样品槽模板（梳子）

6、锥型瓶（或）7100ml50ml、一次性塑料手套

8、凝胶成像系统9［操作步骤］酶切反应体系的建立（口反应体系）

1.201按下表要求配制酶切反应体系质粒DNA6Hl10XBuffer2u1Hindlll1u1H O10P12EcoR I1u1混匀后置37c水浴3ho反应体系的制备（反应体系）

2.PCR25ulDNA2ul10X Buffer

2.5Pl按下表要求配制酶切反应体系dNTP

0.1ul H2O

17.2ulPrimer-

10.1u1TaqE1u1甘油口Primer-

20.1ul50%21混匀后，离心然后按2000rpm30sec,℃945min℃40sec9455℃40sec35cycles72℃90sec72℃3min电泳琼脂糖凝胶（酶切）和琼脂糖凝胶（反应）

3.1%

1.5%PCR将口酶切和产物进行电泳，观察结果201PCRVocabulary inBiochemical technologyAAbsorbanceAbsorbentAbsorptionchromatographyAbsorption spectrumAcrylamide,Aa/AcrAdenine,AAffinity chromatographyAgarose，Agarose gelelectrophoresis AGEAllelespecific oligonucleotide,ASOAmmonium persulfate,APAngle rotorAnionexchange chromatographycCarrierampholyteCation exchangechromatographyCellulose acetatemembrane electro-phoresis,CAME吸光度Cellulose ion-exchangers吸附剂Centrifuge吸附层析吸收光谱Centrifugal force丙烯酰胺（单体）Centrifugal technique腺喋吟Coenzyme亲和层析Column chromatography琼脂糖Cytosine,C琼脂糖凝胶电泳等位基因特异性寡核甘酸杂交D过硫酸胺Denature gradientgel eletro角式转头-phoresis,DGGEDeoxyribonucleic acid,DNA Dialysis载体两性电解质阳离子交换层析Differential centrifugation醋酸纤维薄膜电泳纤维素离子交换剂离心机离心力离心技术辅酶柱层析胞喀咤变性梯度凝胶电泳脱氧核糖核酸透析差速离心EEletro-osmosis不连续聚丙烯酰胺凝Emission spectrumDiscontinuousPAGE胶电泳Exclusion chromatography电渗G发射光谱Gel chromatography排阻层析Gel filtrationGuanine,G凝胶层析H凝胶过滤鸟喋吟Homogenizer Holozymase匀浆器I全酶Ion-exchange chromatographyIon-exchange gel离子交换层析Ion-exchange resin离子交换凝胶离子交换树脂Isoelectric focusing,IEF等电点聚焦Isoenzyme同工酶L配基Ligand溶解温度M载体流动Melting temperature,Tm Matrix相分子筛过滤Mobile phaseMolecularsieve filtration四亚甲基N,N,N,N-N双丙烯酰胺四甲基乙二胺N,N,N/N-methyIencBisacrylamide,BisN/N/N/N-tetramethylethylenedi-amine,TEMED渗透OsmosisP纸层析Paper chromatography分配层析分配系数Partition chromatography光电比色法Partition coefficient质粒聚丙烯酰胺凝胶Photoelectric colorimetryPlasmid聚合酶链反应Polyacrylamide gelelectrophoresis,引物PAGEPolymerase chainreaction,PCRPrimerR相对离心力Relative centrifugalforce,RCF限制性内切酶限制性片段长度多态Restriction endonuclease,RE性Restriction fragmentlength速率区离心法polymorphism,RFLP反向渗透Rate zonalcentrifugation每分钟转速核糖核酸Reverse osmosis转头Revolution perminute,rpm盐溶Ribonucleic acid,RNA Rotor盐析葡聚糖凝胶sSDS-聚丙烯酰胺凝胶Salting in沉降系数Salting out沉降速率沉降时间Saphadex琼脂糖凝胶测序单链构象多态性SDS-PAGESedimentation coefficientSedimentationvelocitySedimentation time,STSepharoseSequencingSingle strandconformativepolymorphism,SSCPSodium dodecylsulfate,SDS分光光度计Spectrophotometer分光光度法Spectrophotometery固定相水平式转头Stationary phaseSwing-out rotorT透层层析Thin-layer chromatography,TLC胸光率咤转腺喀Transmittance转化转染Thymine,T转导位T ransformation薄TransfectionTransductionTranspositionU超滤Ultrafiltration尿啥咤Uracil,U紫外可见分光光度法载体VVisible andultraviolet Spectro-photometeryVector。