Edited by samita, 20 March 2009 - 10:52 AM.
Posted 20 March 2009 - 11:12 AM
genius does what it must
i do what i get paid to do
Posted 20 March 2009 - 11:19 AM
Depends on the species of bacteria. And in some species there is a mix of both.
Posted 26 March 2009 - 07:52 AM
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...
Posted 26 March 2009 - 11:32 AM
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.
Posted 26 March 2009 - 03:59 PM
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.
Posted 26 March 2009 - 04:22 PM
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?
Posted 27 March 2009 - 05:28 AM
(transposon, can be on chromosome and plasmid as well right ? )
hope to hear from you soon, again. thanks in advance !
Posted 27 March 2009 - 08:46 AM
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.
Posted 27 March 2009 - 05:36 PM
and again, thanks for the replies. it helps a lot