Gateway clonning.pdfVIP

1 | Life Technologies Proprietary Confidential | 6/7/2010 Gateway? Cloning ---基因克隆和蛋白表达的有力工具 Heshu Lv Technical Sales Specialist North China, Invitrogen 2 | Life Technologies Proprietary Confidential | 6/7/2010 The Gateway? Cloning System Your Application Gene1 Gene2 Gene3 Gene4 Gene Protein Localization Gene Protein Purification Gene Cell-Free Your Application Gene Gene RNAi Gene Protein Interaction Gene Entry Clone Gene PCR Gene Synthesis ORF Collection Library Your Source 3 | Life Technologies Proprietary Confidential | 6/7/2010 GateWay技术： —用于重组的四个特异性重组位点 attL attR LR reaction LR Clonase BP reaction BP Clonase attB attP 25bp 两种可逆的重组反应 两种重组酶 4 | Life Technologies Proprietary Confidential | 6/7/2010 Gateway?技术 ---含有特异性重组位点的四种载体 at tL 1 attL2 Entry Clone gene at tR 1 attR2 Destination Vector ccdB LR Reaction LR Clonase? Mix at tP 1 attP2 Donor Vecto r ccdB at tB 1 attB2 Expression Clone gene + + Kmr KmrApr Apr BP Reaction BP Clonase? Mix 入门克隆 表达克隆 目的载体 供体载体 5 | Life Technologies Proprietary Confidential | 6/7/2010 Entry Clone L2GeneL1 Gateway? BP Clonase? 1. Gateway? BP Cloning + attB PCR Product B2GeneB1 Donor Vector P2ccdBP1 2. TOPO? Cloning TOPO? PCR Product +TOPO-Activated Entry Vector L1 L2 Gene + digested DNA Fragment GeneB1 digested Entry Vector L2L1 Ligase 3. Restriction/Ligase Cloning 4. Custom/Pre-made Entry Clone Gateway? ORF Collections L2ORFL1 5. Gene Synthesis6. cDNA Library Construction Generate a Gateway? Entry Clone 6 | Life Technologies Proprietary Confidential | 6/7/2010 ? Add the attB1 sequence to the 引物5’端 ? Add the attB2 sequence to the 引物3’端 attB1 5’ - GGGG ACA AGT TTG TAC AAA AAA GCA GGC TNN NNN... attB2 5’ - GGGG AC CAC TTT GTA CAA GAA AGC TGG GTN NNN... Gene Specific Primer Sequence Gene Specific Primer Sequence 通过 PCR 构建入门载体 7 | Life Technologies Proprietary Confidential | 6/7/2010 Generating Gateway? Entry Clones geneattB1 attB2 + attP1 attP2 ccdB Donor Vector KanR 8 | Life Techno