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1、目的基因模板Y1目的基因模板Y2引物分别为：P1,R1;P2,R2一、简易融合PCR1.待融合片段的独立扩增具体步骤：ddH2O9.5ul2×Gcbuffer25ul用量参考TaKaRa目录dNTP8ulPrimer10.5ul(稀释后的浓度为25uM,终浓度0.25umol/L)Primer20.5ul(终浓度0.25umol/L)LATaq0.5ul(产品浓度为5U/ul)总DNA6ul(浓度是0.05ug/ul,质量为0.3ug)50ul体系反应程序95℃5min94℃1.5min65℃30s×3072℃2min72℃7minPCR后得到：目的基Y150
2、uL，目的基因Y250uL。DAN电泳检测样品2.融合PCR反应PCR产物(不回收)等体积混合为模板ddH2O4.5ul2×Gcbuffer25ul用量参考TaKaRa目录dNTP8ulLATaq0.25ul(产品浓度为5U/ul)总DNAxul(浓度是0.05ug/ul,质量为0.3ug)25ul体系ssⅢ、ssⅡ片段(不回收)的加入量依次以0.05、0.10、0.15、0.20、0.25、0.50、1.0、1.5、2.0μL等体积混合(对应的DNA量分别约2、4、6、8、10、20、40、60、80ng反应程序95℃5min94℃1.5min65℃30s×
3、572℃2min继续加入25uL反应溶液（Taq酶，dNTP,buffer，引物G1和G4）继续pcr95℃4min94℃40min65℃45s×3072℃1min72℃10minPCR后得到：取5uL电泳检测，检查是否出现目的基因，切胶纯化一、高效融合PCR1.引物设计大写序列为扩增片段的特异性引物序列,小写序列为相邻片段的互补序列特意序列为20左右，互补序列为202.待融合片段的独立扩增具体步骤：ddH2O9.5ul2×Gcbuffer25ul用量参考TaKaRa目录dNTP8ulPrimer10.5ul(稀释后的浓度为25uM,终浓度0.25umol/L
4、)Primer20.5ul(终浓度0.25umol/L)LATaq0.5ul(产品浓度为5U/ul)总DNA6ul(浓度是0.05ug/ul,质量为0.3ug)50ul体系反应程序95℃5min94℃1.5min65℃30s×3072℃2min72℃7minPCR后得到：目的基Y150uL，目的基因Y250uL。DAN电泳检测样品扩增产物用0.8%的琼脂糖凝胶电泳检测,确定目的条带,切胶GelExtractionMiniKit回收。待融合片段引物反应条件长度(bp)AlAl1,Al235cycles(95bC40s,61bC50s,72bC3min)1593A
5、bAb1,Ab230cycles(95bC30s,61bC30s,72bC4min)1966ArAr1,Ar235cycles(95bC40s,61bC50s,72bC2min)1037BlBl1,Bl235cycles(95bC40s,57bC50s,72bC6min)3159BhBh1,Bh230cycles(95bC30s,58bC30s,72bC3min)1451BrBr1,Br235cycles(95bC40s,58bC50s,72bC1.5min)633ClCl1,Cl235cycles(95bC40s,61bC50s,72bC3min)1593C
6、bCb1,Cb230cycles(95bC30s,61bC30s,72bC4min)1966CrCr1,Cr235cycles(95bC40s,61bC50s,72bC2min)1062ChCh1,Ch230cycles(95bC30s,61bC30s,72bC3min)14291.融合PCR反应测定各回收产物中目的片段浓度,在50Ll体系中加入等摩尔的待融合片段(要求DNA总量不少于800ng),不添加引物,使用PfuDNAPolymerase进行各片段的互补延伸,以形成全长的融合PCR产物(中间产物)。融合产物待融合片段反应条件长度(bp)AlbrA,lA
7、b,Ar11cycles(95bC15s,60bC20s,72bC9min)4.5BlhrB,lBh,Br11cycles(95bC15s,56bC20s,72bC10min)5.2ClbrhC,lCb,CrCh13cycles(95bC15s,60bC30s,72bC10min)5.91.融合片段的全长扩增融合产物全长扩增反应条件融合产物引物反应条件长度(bp)AlbrAl1,Ar230cycles(95bC10s,61bC30s,68bC4min)4.5BlhrBl1,Br230cycles(95bC10s,57bC30s,68bC5min)5.2Clbr
8、hCl1,Ch230cycles(95
