Gateway-Entry-Options-seminar.pptxVIP

The Gateway? Cloning System How to generate an entry clone Contents Options for entering the Gateway? system Defining the BP Clonase? reaction Defining the LR Clonase? reaction Description of Ultimate? ORF collection Description of Vector NTI Advance? software for in silico cloning Invitrogen Proprietary Confidential 2 The Gateway? Reactions Invitrogen Proprietary Confidential 3 Different ways to generate the entry clone TOPO? Cloning TOPO? BP Clonase? BP Cloning Entry Clone L2 Gene L1 4. Pre-made entry clone 5. Custom-made entry clone Ligase Restriction/Ligase Cloning ORF Collection L2 ORF L1 Invitrogen Proprietary Confidential 4 BP Cloning – The Reaction + + BP Clonase? Invitrogen Proprietary Confidential 5 BP Cloning - Primer Design for PCR GGGG and the attB1 sequence must be added to the 5’-primer (sense) GGGG and the attB2 sequence must be added to the 3’-primer (antisense) attB1 5’ – GGGGACAAGTTTGTACAAAAAAGCAGGCTNNN… attB2 5’ – GGGGACCACTTTGTACAAGAAAGCTGGGTNNN… Invitrogen Proprietary Confidential *After overnight incubation BP Cloning – Some Examples Size (kb) PCR DNA (fmol) PCR DNA (ng) Colonies/?l Transformation Correct Clones/Total Clones Examined 0.26 15 38 3 7.5 1223 2815 10/10 1.0 15 38 10 25 507 1447 49/50 1.4 15 38 14 35 271 683 48/50 3.4 15 38 34 85 478 976 9/10 4.6 15 38 46 115 190 195 10/10 6.9 15 38 69 173 30 (235)* 54 (463)* 47/50 10.1 7.5 37.5 50.5 252.5 16 (112)* 42 (201)* 15/16 Tyrosine Kinase Transferrin Receptor Target Template: EIF4e b-Adaptin MAP4 attB-containing primers: + + - - + + - - + + - - + + - - + + - - Platinum? Pfx DNA Polymerase Platinum? Taq DNA Polymerase High Fidelity Platinum? Taq DNA Polymerase Total RNA isolated from HeLa cells, first strand cDNA synthesized using THERMOSCRIPT? RT. BP Cloning – RT-PCR Using attB-Containing Primers Invitrogen Proprietary Confidential 8 TOPO? Cloning -TOPO?TA Invitrogen Propr