Hello all,
I got an e.coli strain that is resistant to speculation, however I do not know the genomic location of this spectR gene. Any easy ways of finding this out or only chromosomal walking would do?
You can fragment the chromosome, self-ligate the fragments to form circles and PCR out of your gene into the chromosomal sequences (divergent primers) and than sequence the resulting product. However, these days it will probably be easier to generate an NGS library of the fragmented chromosome and sequence the whole thing.
Dear Sivan,
I think the simpliest thing to do would be to digest the bacterial DNA with the insert of your gene with a frequent cutter restriction enzyme that cuts near one end of your gene (for example, 100-200bp from the end, and presumably only a few hundred base pairs from the integration site in the bacterial genomic DNA), then ligate a linker to the digested DNA fragments. Now you can perform PCR using a primer specific to the linker primer and a primer specific to the 100-200bp sequence of your gene. The PCR reaction should amplify DNA sequence between the linker and sequence of the 100-200bp of your gene. By sequencing the PCR fragment, you should find the junction of your gene sequence and the bacterial genomic DNA, which should define the site of integration. Let me know if this makes sense to you.
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