5 replies to this topic

[Lefty]

Was wondering why you would prep Chromosomal DNA instead of just prepping plasmid?  If you have nothing but chromosomal DNA and need to prepare more, do you go about it the same way as if you have plasmid (Transformation/Streak/Pick Colony and innoculate media/miniprep)?

Thanks

[prabhubct]

Chromosomal DNA could be amplified using PCR. You could treat it as insert for cloning to amplify by cloning.

Chromosomal DNA is linear it is prone to degradation by exonucleases. Again may be origin of replication for chromosomal DNA may not be as efficient as plasmids ori.

[Lefty]

So, I can just use as I would as if I had a plasmid prep?

[bob1]

Not exactly the same - plasmid preps use an alkaline lysis system that selects preferentially for circular forms of the DNA, and damages other forms.  If you want a decent prep of chromosomal DNA you need to do something like a standard phenol-chloroform.

Plasmids are used by the bacteria as a different system of storing DNA, that can be shared and easily replicated - the chromosomal DNA is not so easily manipulated and contains elements essential for the viability of the bacterium.

[Lefty]

Thanks for the replies... I guess what I am asking, is if I can use the sample of chromosomal DNA that I have the same way as I would if I just had a sample of plasmid DNA?  Meaning, can I use the chromosomal DNA the same as I would plasmid DNA for a transformation?  (Not asking if the methodologies for prepping each one is the same... ie Qiaprep vs. Qiamp kits)

Thanks again

[Trof]

Chromosomal DNA doesn't contain signals for replicating inside other cell, nor the shape or size to make this possible. If a bacteria is a copy-center house and plasmid is a piece of paper someone wants to multiply, you put the paper to copy machine and make many copies. Then demolish the house and get the papers you want out.
But you can't put a chromosomal DNA into a copy machine, because it doesn't fit there, chromosomal DNA is like a huge plan for a different house.

Houses can replicate on their own, but in a different way. But you need a complete house to do that, not only a plan.
The best way to get more chromosomal DNA is to grow again whatever the DNA is from. Also, isolated DNA would be fragmented, it's not even intact now and can't even replace some other cell original chromosome. You can however amplify the whole chromosomal DNA in a PCR manner (in vitro), by some whole genome amplification kit. But in comparison to plasmid cloning, this would be AFAIK far too more expensive.