**4.7 Selection of positive recombinants**

28 Bacterial Artificial Chromosomes

We found that for the initial electroporation of BAC DNA into recombineering bacterial strains, a "dirty" prep of BAC DNA containing genomic DNA prepared by alkaline lysis

With "Quick Prep" BAC DNA (described above), electroporation can be performed at 1.8KV with a time constant of 5µs. Cells are then resuspended in 1ml LB media and incubated for 1hr at 30C. As with standard practice, 100ul cells are streaked on selection plates and incubated at 30C overnight. To the remaining electroporated cells, 4ml LB medium are added and incubated overnight at 30C. The following day, cells were plated on selection

Electrocompetent cells are prepared by inoculation of 50 ml LB plus appropriate reagent to select for the BAC clone of interest at 1:50 dilution from an overnight culture. Cells are incubated at 30C until the culture achieve**s** an OD600 of 0.50, which is usually 3-4 hrs. Cells are then divided into two aliquots, one for induction of lambda phage proteins and one un-

To induce the lambda phage proteins, cells are incubated at 42C for 15 minutes, and then harvested by centrifugation at 5000xg for 15 minutes at 4C. Cells are then washed 3 times in 25ml ice-cold 0.2µm-filtered ddH2O containing 10% glycerol, resuspended in 30µl 10%

Recombineering modifies the genomic locus using a linear targeting construct. The linear targeting construct can be generated either by PCR or excision from plasmid DNA, as

A convenient method for generating linear targeting vectors is via a PCR based approach in which primers are designed against two important sequence elements. The 5' end of the primer contains sequence homology to the targeted locus while the 3' end contains sequence for PCR amplification of the targeting construct. When these primers are used in a PCR reaction, the product produced contains both homology to the target and the targeting construct. Using a PCR based approach allows an investigator to use the same template to target an alternative locus. By changing the 5' homology of the primers to match an alternative locus, a new linear targeting construct can be generated. Therefore, the template

Cloning longer (100-500 bp) homology arms into the targeting construct is an alternative approach to generating a linear targeting vector. Using this type of construct is also useful for cloning sequence from BAC DNA into high copy plasmids for subsequent modification and use for in vitro cell targeting (i.e. embryonic stem cells). The linear

glycerol in ddH2O, and then transferred to cooled cuvettes for electroporation.

alcohol precipitation was sufficient.

**4.5 Generating a targeting vector** 

**4.5.1 PCR based approach** 

**4.5.2 Plasmid based approach** 

induced control.

discussed below.

plates and transformed cells are obtained from this selection.

used for generating the linear targeting construct is modular.

**4.4 Preparation of induced electrocompetent cells** 

Selection of positive recombinants can be facilitated by inclusion of a PGK-neomycin selection cassette in the targeting vector that confers kanamycin resistance to recombinant clones. Cells are plated on LB agar plates containing 15ug/ml kanamycin and grown at 30C overnight, as described above. Following antibiotic selection, clones are screened for correct targeting using DNA isolated from kanamycin resistant clones in a PCR reaction using two different sets of primers that would amplify a product across the 5' and 3' recombination sites. PCR reactions are then analyzed on 1% agarose gel to correctly targeted clones. The PCR products are then sequenced to ensure the recombination had taken place as expected. Southern Blot analysis is another method for identifying the inclusion of the targeting construct into the BAC. While Southern Blot is sensitive, it gives no information on the sequence and requires additional time and labeling reagents (i.e. radioactive nucleotides).
