Plasmidic or chromosomic gene?

[shonlaanh]

Hi all,
I am looking for the protocol of determining gene location (in plasmid or chromosomic). Thanks for your direction

[aftabac]

well
if you looking for the gene on the plasmid DNA or chormosomal DNA, then first you have to determine the location of the gene. for this it is very easy if it is some marker gene. i.e. metal resistance or antibiotic resistance gene. so if your gene of interest is some metal resistance gene or antibiotic resistance gene, then simple you can isolate Plasmid DNA from your strain and transform it to cloning strain like DH5a or C600 or any other and grow that to metal or antibiotic, if it grew then it mean the gene is located on plasmid.
or other way you can cure your plasmid from strain by any of the given procedure and grow that on metal or antibiotic.
in this way you will find the location of the gene. i.e. whether it is located to plasmid or chormosome.
then you can further narrow down the location by fragmenting the plasmid or chormosomal DNA by restriction enzymes (rare cutter) and clone the DNA pieces and then transform to cloning strain and then isolate for your marker.i.e. metal, antibiotic or some nutriotional marker.
so in this way you can plan the stratagy and can work to locate your gene.
best of luck for your work

regards
aftab

[shonlaanh]

Thank you, aftab.
Actually i am working with a wild type strain, which doesnt have a selection marker such as metal, nutritional or antibiotic resistance.
The strain produces a bacteriocin (a peptide antibiotic) so it also has a bacteriocin immune gene in the cluster. How should i go?

[aftabac]

Hi
welcome
well, what type of gene is under your investigation. is it some structural protein or an enzyme gene or some other??

regards
aftab

[shonlaanh]

Hi aftab
I am studying gene encoding for bacteriocin (peptide <10KDa). It might occur in plasmid or chromosome or both.

[aftabac]

well
then it is simples as i am sure that bacteriocin gene is sequenced so you can design the perimer and can amply the gene in a way that first from total DNA, then from chormosoamal DNA and also from Plasmid DNA, so you perform PCR from all these samples and you can confirm where it is located.
then you have to find out the location of your gene, and you can easily FISH (Flourecence insitu hyberidization) out the location of your gene by using specific probes.
you can also use another approach in which you can gigest the chromosomal and Plasmid DNA by restriction enzymes separatly and then can find out where you gene is located by perform southern bloting.
anyhow
best of luck for you
regards
aftab

[shonlaanh]

It is clear to me now.Thanks a lot, aftab.