3 replies to this topic

[Baroudeur]

Does anyone have protocols (not kits) for the extraction of plasmid DNA and genomic DNA from bacteria ? and at what level is the difference?

Thx

[perneseblue]

The main difference is the pH of the denaturation solution.

pH of plasmid denaturation solution is about 12.1-12.3. At this pH all DNA is denatured, and upon renaturation only plasmid DNA can renature to form dsDNA. (as a plasmid are supercoiled rings of DNA -and thus are entangled)

pH of genomic DNA denaturation/extraction solution is about 7-9 (depending on the exact protocol you are using). At this pH large molecules of DNA are not denatured, though there maybe some breathing.

[pDNA]

i really like the CTAB protocol for the extraction of genomic DNA ...it really gives you high quality gDNA for e.g. sequencing projects. You can find an example protocol here. There are better and more detailed protocols in the web ...but this was the one i was able to find quickest.

for plasmid DNA extraction you should go for the method of Birnboim and Doily (1979). You can find the protocol here.

Hope this helps!

Regards,
p

[Baroudeur]

Thank you guys, that was very helpful. In my lab we actually use a phenol:chloroform:isoamylalcohol extraction protocol that omits CTAB, I once tried to make the solution but the product wasn't available. So I guess I should either buy it or borrow some from neighbours. And I guess the main problems I am facing in genomic DNA extraction are at the lysis phase because there are so many protocols out there for lysis (SDS, TE, Lysosyme, no lysosyme, proteinase K, 37°C, 50°C .... ) so I'm kinda lost and still trying to make out my own mod based on all this ... As for plasmid extraction, thanks perneseblue, that was so enlightning.

Baroudeur