Genomic DNA - (Mar/20/2009 )
Does genomic DNA from bacteria is linear or circular
genomic dna from e. coli is circular.
samita on Mar 20 2009, 10:51 AM said:
Depends on the species of bacteria. And in some species there is a mix of both.
i am somehow confused by the fundamental basic of bacterial Genomic DNA and plasmid DNA.
when we're saying genomic DNA extraction using PCI, CIA method (Ausubel et. al.) ,
are we ONLY extracting the chromosomal DNA ?
coz i read from somewhere, saying : "Plasmid DNA isolation is more demanding than genomic DNA isolation because plasmid DNA must be separated from chromosomal DNA, whereas a genomic DNA isolation needs only to separate total DNA from RNA, protein, lipid, etc."
the bold part, genomic DNA isolation needs separate total DNA...
so means total DNA is what we extracted in the end, means, despite chromosomal DNA, and we would get mitochondrial DNA, plasmid DNA etc since it is total genomic ?
if that so, why would we need particular plasmid DNA extraction then ? just to prove the gene is located on plasmid ?
i m confused. i need someone to help me back to the right track...
When we do a genomic DNA extraction, we actually do is a total DNA extraction. All DNA in the cell is extracted. However since most DNA in the cell is genomic DNA, the term tends to stick (even if the sample contains mitochondrial DNA, or plasmid DNA).
So why do we have plasmid extraction protocol?
Often we are only interested in the plasmid. We are have no interests in the DNA of the host that the plasmid grew up in. The presence of host genomic DNA is also undesired as it would act as a contaminant to molecular biological assays. For example you can't do a plasmid ligation if the vector is contaminated with genomic DNA. The vector would just ligate to the contaminating DNA instead. Contaminating host genomic DNA would also become background that would obscure assays such as restriction digest. You won't see your bands if it is overlaid by a smear.
The plasmids are often amplified so that we have enough DNA to use for a transformation in another organism, ie to introduce a new gene, capture a segment of DNA, add a specific DNA sequence at a specific loci in the genome. Often there is only so much DNA you can transform or micro-inject into an oocyte. Thus it is desirable that the DNA being transformed be pure.
Lastly you want to be certain that the effects you are seeing is caused by DNA that you have introduced and not the random bit of genomic DNA.
orite thanks for ur reply.
bcoz i m now screening a gene from a bacteria, and i m uncertain whether the gene is located on chromosome or plasmid; and i get the desired PCR band size using the chromosome and plasmid DNA as my template and thus confusing me.
if it's as like u said, the genomic DNA extraction does contain plasmid DNA inside, then i think the PCR from plasmid DNA could be used to further confirm the gene is actually located in plasmid ? pls correct me if i m wrong.
hope to hear from you soon. thank you.
tyrael on Mar 26 2009, 03:59 PM said:
hope to hear from you soon. thank you.
Yes, provided that your PCR amplifies across the junction between the gene and the plasmid. This PCR fragment must be unique to the plasmid.
However the PCR is does not definite proof. The plasmid could be intergrated into the genome of the bacteria.
I think a southern blot would be a good second screen once you have identified several isolates that you believe have the gene in the plasmid or genome. The southern blot which would be more definitive.
Do you know anything about the sequence or size of the plasmid?
my work is to screen for particular gene from bacteria isolated on my own... so i dont know whether the bacteria isolated, will possess the gene on chromosome or plasmid... (i dont even know the bacteria will have the plasmid either...even if it does, what could i do to get to know what plasmid inside it? what i mean is, a bacteria can obtain different plasmid, right ? ) from the research journals that i went through, some have proved that the gene that i looked for is located on chromosome, some said on plasmid, and even on transposons... which makes me headache now.. =(
(transposon, can be on chromosome and plasmid as well right ? )
hope to hear from you soon, again. thanks in advance !
how about a pulse field gel electrophoresis using lysed bacteria in gel plugs?
That way you can isolate intact genomic chromosome and plasmids. If you then do a southern blot you can determine if you gene is on the chromomsome or plasmid.
OK. i will give it a try then.
and again, thanks for the replies. it helps a lot