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Gateway® Technology
Version E
13 May 2010
25-0522

Gateway® Technology
A universal technology to clone DNA sequences for
functional analysis and expression in multiple systems
Catalog nos. 12535-019 and 12535-027

A Limited Use Label License covers this product (see Purchaser Notification). By use of this
product, you accept the terms and conditions of the Limited Use Label License.ii

Table of Contents
Table of Contents ..................................................................................................................................................... iii
BP and LR Recombination Reaction Protocols for Experienced Users ............................................................. v
Kit Contents andStorage....................................................................................................................................... vii
Accessory Products .................................................................................................................................................. x

Introduction ......................................................................................................................1Overview ................................................................................................................................................................... 1
The Gateway® Technology ...................................................................................................................................... 3
Gateway® BP and LR Recombination Reactions.................................................................................................. 5
Features of the Gateway® Vectors .......................................................................................................................... 8
Gateway® Nomenclature......................................................................................................................................... 9

Methods...........................................................................................................................10
Options to Create Entry Clones ............................................................................................................................ 10
Designing attB PCR Primers................................................................................................................................. 12
Producing attB-PCR Products............................................................................................................................... 15
Purifying attB-PCR Products ................................................................................................................................ 16
Creating EntryClones Using the BP Recombination Reaction ........................................................................ 17
Performing the BP Recombination Reaction....................................................................................................... 20
Transforming Competent Cells............................................................................................................................ 23
Sequencing Entry Clones....................................................................................................................................... 26
Creating Expression Clones Using the LR Recombination Reaction .............................................................. 27
Performing the LR Recombination Reaction...................................................................................................... 29
Constructing a Gateway® Destination Vector..................................................................................................... 31
Troubleshooting...................................................................................................................................................... 37

Appendix...