I just can't extract any DNA out of my B.subtilis culture? Why?

Refering to Cheng, H.R., and Jiang, N., (2006) "Extremely rapid extraction of DNA from bacteria and yeast", Biotechnology Letters, 28: 55-59.


I followed most of the protocol, except that I used P:C:I (25:24:1) instead of tris-saturated phenol.


1. 1ml of culture, centrifuged, washed with STE buffer twice.


2. Resuspend cell pellets with TE buffer


3. Add 1x volume of P:C:I (25:24:1), vortex for 60secs.


4. Centrifuge at max speed for 5mins at 4C.


5. Transfer aqueous layer to new tube, mix with 1x volume of chloroform, centrifuge at max speed for 5mins at 4C (repeat until no white interface is present)


6. Transfer aqueous layer to new tube, add RNase A, incubate at 37C for 30mins


7. Add 1x volume of chloroform, mix and centrifuge at max speed for 5mins.


8. Transfer aqueous layer (which theoretically should contain pure DNA) to new tube and store at -20C


Before RNAse A treatment, I can see a %26gt;10kb band n trace of RNA, however after the treatment, no band was seen. Why?

I just can't extract any DNA out of my B.subtilis culture? Why?
Either i) your cells are not lysing, ii) you are losing genomic DNA at step 3 (I am not a fan of doing the phenol and choloroform (PCI) at the same time, you save very little time doing this), iii) you are losing the DNA in step 5 (you need to make sure you transfer all of the aqueous layer and I often end up taking a very small amount of the white bottom layer also into the next step), or iv) your RNase is contaminated with DNase. I would try the extraction without the RNase step and see what you get. Also, are you digesting the DNA before gel electrophoresis (you would see a smear), and are you doing an ethanol precipitation at the end and reconsituting the DNA in buffer? If you are adding DNA from the chloroform extraction step directly to the agarose gel, you may be losing significant amounts at that step also.





Good luck!
Reply:Are you looking for RNA or DNA? At the end, you state that the *RNA* band was gone after RNAse treatment - which is not too surprising ;-)





If, however, you mean that your DNA weas gone after RNAse treatment: are you sure your RNAse isn't contaminated with any DNAse?


Also - the DNA is insoluble in the chloroform, so that final purification stage is just to remove the RNAse. Will you be needing RNAse-free DNA subsequently? If not, then you could try missing that stage out.





Only things I can think of off the top of my head.


Good luck.

love song