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Targeted mutagenesis using a linear fragment

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#1 cerqueiragm



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Posted 14 June 2011 - 03:26 AM

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.

Edited by cerqueiragm, 14 June 2011 - 03:29 AM.

#2 phage434



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Posted 14 June 2011 - 03:46 AM

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.

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