Acid phenol extraction seems to be feasible in my lab (A/c to the facilities available). I could not find out the protocol to fish only the supercoiled form of plasmid. Please shed some idea.
Thank you
Laurence Stuart Dawkins-Hall
Dear Balaji
I cannot comment on supercoiled isolation as a specific fraction but what I will say is that you SHOULD not use acid phenol:
- Acid phenol is designed for purification of RNA and your DNA will get sequestered and lost in the interface
- For extraction of all types of DNA use Tris saturated (pH 7.5) phenol
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Sibnarayan Datta
You can isolate plasmid DNA following the classical alkaline lysis protocol, followed by agarose gel electorphoresis of the preparation, then slice out the supercoiled fraction and purify it using a gel purification kit. This way you can get plasmid DNA, most of which will be in supercoiled configuration
best wishes,
SD.
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Laurence Stuart Dawkins-Hall
I agree with Andrew:
- I am old enough to remember the days when DNA was extracted manually and maxipreps were synonymous with CsCl ultracentrifugation
- Apart from the fact that you will derive the cleanest DNA you will ever purify, it separates according to conformation: upto 3 seaprate bands under UV, one of which you can extract with a syringe and will be supercoiled
- Good suggestion Andrew: As somebody who has done about 100 CsCl purifications wish I d tyhought f my past !! Definitely the best method although you will need access to an ultracentrifuge that spins over night at least 50,000 rpm
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