Concepts behind plasmid prep
Posted 24 March 2010 - 07:21 AM
Posted 24 March 2010 - 08:34 AM
Eum I know this sounds strange, a picture might explain it better.
The circles are plasmids, the "lines" are chromosomal DNA.
I hope you understand it?
So in short: you denature both chromomal DNA and plasmid DNA and then you renature it with the acid. And only the plasmid DNA can renature effectivly.
Edited by pito, 24 March 2010 - 08:37 AM.
Posted 24 March 2010 - 09:21 AM
Hello, I was curious to know why a plasmid prep isolates only the plasmid DNA and not the bacterial chromosome? Could someone explain how the different steps of a typical plasmid prep lead to the isolation of only plasmid DNA?
To put it another way:
With the most commonly used method (alkaline lysis) chromosomal and plasmid DNA are separated by differential denaturation/renaturing. Bacteria are lysed with ionic detergent (SDS) and NaOH. During lysis both types of DNA are denatured. Subsequent rapid neutralization (by addition of a concentrated potassium acetate buffer) allows only the covalently closed plasmid DNA to reanneal and stay solubilized. Most of the chromosomal DNA and proteins precipitate in a complex formed by an insoluble potassium-SDS complex. This is removed by centrifugation.
Further purification by anion-exchange (eg Qiagen columns) removes further contaminants such as salts and protein. This relies on selective binding/elution of the strongly negative charged DNA to a gel containing positively charged chemical groups.
Hope this helps.
Edited by klinmed, 24 March 2010 - 09:23 AM.