IDT
IDT|
Red®/ET® Recombination can be applied to numerous DNA engineering tasks.
The following example shows the construction of a conditional knockout targeting vector for the murine homeobox protein Hox-A11 using the BAC Subcloning Kit and the Quick & Easy Conditional Knockout Kit (loxP/cre).
1. PCR Homology regions are added to the linear minimal vector by PCR amplification using vector-specific primers containing 50 bases homologous to Hox-A11 at the 5 end and Phusion High-Fidelity DNA Polymerase. 
2. Red/ET Recombination Starting from a BAC containing > 100 kb of genomic DNA from mouse chromosome 6 a 15239 bp fragment is subcloned into the minimal vector by Red/ET Recombination using the BAC Subcloning Kit. 
3. Red/ET Recombination To insert a single loxP site a floxed PGK-gb2-neo cassette containing 50 bp Hox-A11 homology arms added by PCR amplification using Phusion High-Fidelity DNA Polymerase is introduced at the position of choice by Red/ET Recombination using the Quick & Easy Conditional Knockout Kit loxP/cre. 
4. Excision by Cre Cre-mediated excision to leave a single loxP site behind. (See also our loxP selection cassettes and Cre expression plasmids & strains). 
5. Red/ET Recombination A second floxed PGK-gb2-neo cassette containing 50 bp Hox-A11 homology arms added by PCR amplification using Phusion High-Fidelity DNA Polymerase is introduced at the position of choice by Red/ET Recombination using the Quick & Easy Conditional Knockout Kit (loxP/cre) to yield a targeting vector for ES cell recombination.  |
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