DNA extration from Bacteria spores - (Feb/08/2010 )
HomeBrew on Feb 10 2010, 12:33 AM said:
When I used to work in Bacillus (anthracis, thuringiensis, subtilis, etc.), we used to do a modified version of Kado and Liu (J. Bacteriol. 145:1365-1373) as is described here. It was quite effective.
May I ask why you don't grow the spores and extract the DNA from vegetative cells? Also, you mention that "it is not so easy to get spores" -- can't you just prepare more from vegetative cells by allowing them to sporulate?
May I ask why you don't grow the spores and extract the DNA from vegetative cells? Also, you mention that "it is not so easy to get spores" -- can't you just prepare more from vegetative cells by allowing them to sporulate?
> The bacteria we are working with is an obligate parasite. We collect spores because earlier stages are difficult to harvest. The spores can be washed and isolated easier from possible (host) contaminants.
> Thanks for the citations! - In fact , we used a similar phenol-chloroform method and that's why we think the problem lies in an earlier step i.e. the opening of the spores.
-Mafalda-
Mafalda on Feb 10 2010, 06:25 AM said:
The bacteria we are working with is an obligate parasite. We collect spores because earlier stages are difficult to harvest. The spores can be washed and isolated easier from possible (host) contaminants.
Aha -- I asked this question gently because I knew there had to be a valid reason you were dealing with spores and that they were hard to get...
Mafalda on Feb 10 2010, 06:25 AM said:
Thanks for the citations! - In fact , we used a similar phenol-chloroform method and that's why we think the problem lies in an earlier step i.e. the opening of the spores.
Prior to the phenol:chloroform step, the 60C incubation in SDS/NaOH/sucrose/Tris followed by digestion with pronase (or proteinase K) was critical. If you use proteinase K, that digestion can be performed at 60C also. You might need to jack up the heat and or time of incubation in the lysis step prior to pronase/proteinase K digestion -- as I recall, some people would even use an autoclave.
-HomeBrew-
HomeBrew on Feb 10 2010, 02:33 PM said:
Prior to the phenol:chloroform step, the 60C incubation in SDS/NaOH/sucrose/Tris followed by digestion with pronase (or proteinase K) was critical. If you use proteinase K, that digestion can be performed at 60C also. You might need to jack up the heat and or time of incubation in the lysis step prior to pronase/proteinase K digestion -- as I recall, some people would even use an autoclave.
> Thanks I will give it a try! Is there a possibility to check if there is DNA after the incubation prior to the phenol:chloroform step?
-Mafalda-
I suppose you could try running a bit of the lysate on an agarose gel and staining it with etidium bromide -- I'm not sure what you'll see, though.
-HomeBrew-