How does plasmid DNA miniprep kit get rid of chromosome DNA? - (Jun/21/2005 )
I'm not good at English writing and hope you understand.
When I use miniprep kit to purify plasmid DNA, how does it get rid of cell chromosome DNA? Which solution separates them and how does it work?
Of course I don't want chromosome DNA in my supernatant, but I just want to know why. What's the difference between these two during lysis process?
I have been having some problems with the alkaline lysis method and have looked up a few different protocols to compare to our labs' method. Here is a really good link that explains each step of the miniprep.
Read steps 3, 4 and 5 under the "Plasmid Preparation" section, for your answer. I'm at work so can't elaborate, sorry.
Hope this helps you,
In a standard plasmid isolation, such as miniprep kits. You go through the following steps:
1/ Pellet bacteria
2/ Resuspend bacteria (resuspension buffer/buffer 1/buffer P1)
3/ Lyse bacteria (lysis buffer/buffer 2/Buffer P2)
4/ Neutralisation Step (neutralisation buffer/buffer 3/ Buffer N3)
When you add buffer 3 it causes the precipitate to form this contains bacterial debris, proteins, and genomic DNA. You remove this by spinning it out and then just using the supernatant which should contain your plasmid DNA.
Hope this helps
During the lysis step, you are also reminded not to agitate/shake your tube so that you don't shear the genomic DNA. The genomic DNA remains attached to the cell wall which will pellet out while the plasmid DNA remains in the supernate.
Well, I don't know about the kit you re using and I feel there is no separate solution in any kit for this genomic DNA removal, but what I say is
after the addition of SDS/NaOH containing solution,
the cell is reptured and cellular content will be there in the solution, these cell debris will be settled while the course of centrifugation,
the addition of potassium acetate brings down the pH of the solution slowly (as it was raised to many folds as NaOH was added earlier), in the elevated pH both genomic DNA and plasmid DNA were turned to single standarded and the this mild pH lowering effect of the potassium acetate makes them reanneal to the original form.
During this phase, as the genomic DNA as it got huge mass when compared to plasmid DNA it takes so much time to reanneal, so while centrifugation, these genomic DNA were separted and settled at the bottom, while plasmid DNA still stays in supernatent.
have a nice time