For all types of BAC modifications.

• Modify any type of bacterial artificial chromosomes (BACs) within 1-2 weeks by using a kanamycin/neomycin cassette

• Optimized for basic modifications such as insertions or deletions of fragments in any type of bacterial artificial chromosomes (BACs) leaving a selectable marker gene

• Also applicable for modifications of bacterial chromosomes or common ColE1 origin plasmids

• High Red/ET efficiency plus convenient removal of the Red/ET recombination protein expression plasmid pRedET (pSC101-BAD-gbaA) after recombination


Description

This kit is designed to modify BACs with Red/ET recombineering technology - a powerful tool for introducing modifications such as insertions and deletions into any type of BAC within 2 weeks.

Red/ET recombineering uses in vivo recombination and proofreading activity to minimize unwanted mutations in your construct. Included in the kit are all the necessary contents for modifying BACs and all materials needed to perform a control experiment intended to familiarize you with this novel technique.

Additionally, a vector converting any E.coli strain into competent strains for application with Red/ET recombineering is provided. pSC101-BAD-gbaAtet comes with a tetracyclin resistance cassette. This cassette contains a temperature sensitive origin of replication (ori) that can only propagate at 30°C - at 37°C the plasmid will be lost. This allows for simple removal of the plasmid when it is no longer needed.

To introduce a mutation (i.e. a selection cassette like the neomycin resistance cassette included with this kit) you first have to introduce homology sequences to the selection cassette. This can be done through a simple PCR reaction.
These short homologous sequences (50 nt each) can be freely chosen for your experimental needs. Simply choose the location to be modified and insert the selection cassette directly at the desired nucleotide with the Red/ET recombineering technology. The PCR product then needs to be electroporated in bacteria with the induced Red/ET recombineering system.

After successful Red/ET recombineering the modified DNA can be recovered using conventional DNA mini-preparation methods.



Overview of one-step insertion of a selectable marker gene

1. Transformation of BAC host with Red/ET
In a first step the Red/ET plasmid pSC101-BAD-gbaA is transferred into the E.coli host that contains the BAC. 



2. BAC Modification by Red/ET
The expression of genes mediating Red/ET is induced by adding L-arabinose and a temperature shift from 30°C to 37°C. After induction the cells are prepared for electroporation and the PCR product with the added homology arms is electroporated. 



3. Selection & Extraction of the modified BAC DNA
Select for the antibiotic resistance conveyed by the selectable marker in the PCR product to identify colonies carrying successfully modified BACs. A subsequent DNA mini preparation is used to confirm the desired BAC modification. 




Applications

• Simple BAC modification like insertion of a selection marker, deletion of a fragment with a selection marker gene, insertion of a function fragment together with selection marker gene

• Also applicable for bacterial chromosome modification



Contents

- Red/ET recombination protein expression plasmid pRedET (pSC101-BAD-gbaA); any E. coli strain can be made Red/ET proficient by transformation with this plasmid

- BAC host E.coli strain DH10B already carrying the Red/ET plasmid

- Tn5-neomycin/kanamycin resistance template to be used for your own experiments

- Positive controls to introduce a Tn5-neo/kan cassette in a 150 kb BAC

- Detailed protocols, descriptions of plasmids, maps and sequences


This product is only available to academic researchers, not to commercial sites. Academic customers accept the free license conditions (see link below) by opening the product box.