Bacterial DNA, where does it go? - where does the DNA from bacteria go? (Jul/13/2005 )
When using E-coli for transformation to obtain more plasmid of interest (mini, midi, maxi) where does the bacteria's DNA go? why does it not get "extracted" along with the transformed DNA?
Plamid DNA is small 3-12 kB in general. Bacterial genomic DNA is large... so when you add the lysis buffer to the bacteria all the genomic and plamid dna is released from the cell. Then add the K Acetate and it surrounds the genomic DNA and proteins and carbohydrates and causes them to precipitate (assuming you have been gentle and not sheared the genomic dna by vortexing). When you spin, the genomic dna is precipitated and the small plasmid dna is not.
Nice explanation about genomic DNA precipitation in PLasmid prep....
Thank you for the nice explanation!
U should Know one thing that is only plasmids has a capacity to renature. That is when u Disturb the genomic DNA it cann't becomr renature, But plasmids can.
so that only U can get DNA ( translated plasmid ).
It is also important to gently lyse the cells, so the tiny plasmids can leak out, but the bulky chromosome remains trapped in the membrane shell. This is really the basis of chromosomal separation from plasmid DNA -- the potassium acetate precipitation step causes the cellular debris (with the trapped chromosomal DNA) to precipitate, while the plasmid DNA remains in solution.
If you want to see how important gentle lysis is, vortex your cells at the lysis step -- you'll see plenty of chromosomal DNA...
BTW, the term "genomic DNA" includes the plasmids...