Download TOPO vectors

Table of Contents
Table of Contents ....................................................................................................................................................... iii
TOPO® Cloning Procedure for Experienced Users ................................................................................................ v
Kit Contents and Storage .........................................................................................................................................vii
Accessory Products .................................................................................................................................................... x
Introduction ....................................................................................................................... 1
Overview...................................................................................................................................................................... 1
How Directional TOPO® Cloning Works ................................................................................................................ 3
Experimental Outline ................................................................................................................................................. 4
Methods.............................................................................................................................. 5
Designing PCR Primers ............................................................................................................................................. 5
Producing Blunt-End PCR Products ...................................................................................................................... 10
Setting Up the TOPO® Cloning Reaction............................................................................................................... 11
Transforming One Shot® Competent E. coli .......................................................................................................... 13
Analyzing Transformants........................................................................................................................................ 16
Guidelines to Perform the LR Recombination Reaction...................................................................................... 18
Guidelines to Perform TEV Cleavage of Recombinant Proteins ........................................................................ 20
Troubleshooting .............................................................................................................. 21
Appendix .......................................................................................................................... 23
Performing the Control Reactions .......................................................................................................................... 23
Gel Purifying PCR Products.................................................................................................................................... 26
Map and Features of pENTR™/D-TOPO®............................................................................................................. 28
Map and Features of pENTR™/SD/D-TOPO® ..................................................................................................... 30
Map and Features of pENTR™/TEV/D-TOPO® .................................................................................................. 32
Recipes........................................................................................................................................................................ 34
Technical Service....................................................................................................................................................... 35
Purchaser Notification ............................................................................................................................................. 36
Gateway® Clone Distribution Policy...................................................................................................................... 38
Product Qualification ............................................................................................................................................... 39
References .................................................................................................................................................................. 40
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