Targeted mutagenesis using a linear fragment - (Jun/14/2011 )

Hi everyone,
today I come across with some trouble to UNDERSTAND some gene replacement results.

I've been trying to obtain a knock out bacterial mutant by replacing my gene of interest for a KmR cassette, ok!?. The difference is that I have constructed a linear cassette, which contains the KmR cassette flanked by regions homologous to the flanks of the native gene to be replaced (Flank1 - KmR - Flank2).

I've then transformed the bacterial strain using the linear cassette and obtained Km resistant colonies. Then I've patched them to certify that they are really Km resistant. After this I PCR them using the following primers: Flank 1 forward and Flank 2 reverse (the same used to construct the cassette).

PCR Results:
1. No DNA rxn - No amplification
2. Wild-type rxn - 4 kb band
3. Cassette control rxn - 2 kb band
4. Colony 1 rxn - two bands (4 and 2 kb)
5. Colony 2 rxn - one band (2 kb).


My doubts:
1. Colony 1 could correspond to a single cross-over mutant?
2. Colony 2 could correspond to a gene replaced (knock out) mutant?
3. I've propagated the colony 2 for two more times after initial obtaining. For transformation I've used 10 micrograms of my LINEAR cassette. There is the possibility that the low 2 kb band be due to traces of the linear cassette or you guys think it could be really a double cross over mutant?
3. Someone knows a link to a schematic of a single crossover event using a LINEAR cassette? I tryed everywhere but couldn't find a scheme or diagram.

People, thank you very much for your help.
Kind regards.

I'd say your 4kb band with wt indicates that you need to optimize your pcr reaction. To really verify your clone 2, I would do an inverse pcr, which amplifies the region surrounding the insert. This is done by making primers facing outward from your cassette. Then, you extract genomic DNA, cut with a frequent cutter (Sau3AI, e.g.) then religate at low concentration. This makes a circular fragment containing your cassette and some of the flanking DNA on both sides. Use the ligation product as a pcr template for your outward primers, and amplify the region surrounding your insertion site. Sequence the pcr product to determine the site of your insertion.