Gene Bridges 
 

Home | Company | Distributors | Contact

Applications & Showcases

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.

1.Step: PCR

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.

1. PCR

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.

1. PCR

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).

1. PCR

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.

1. PCR



Red/ET Training Courses

Next course: date to be announced
Details

Download Red/ET Brochure

Red/ET Brochure Gene Bridges' Red/ET Recombination brochure provides a comprehensive introduction to the technology and an overview on Red/ET related products and services.

Japanese Version

Publications

References

 

 

 

Terms & Conditions | Disclaimer | Top of Page
© 2017 Gene Bridges GmbH