Genomic DNA purity...??? - strange 260/280 and 260/230 ratios (Apr/04/2011 )
Hello everyone!
I have a problem and need your help!
In our lab we tried to extract genomic DNA from lung epithelial cells (A549) with the phenol:chloroform protocol.
I thought that everything was under control until we measured the optical density of the final DNA solution.
This is what we had:
260nm = 0.155
280nm = 0.132 (260/280=1.17)
235nm = 0.044 (260/235=3.52)
I think that the above ratios should be ~1.8 in pure DNA.
Do you have any idea what went wrong here? Is this an evidence of contamination? And if yes, what kind of contamination it could be?
I would appreciate every answer! Thank you soooo much! 
Did you extract with chloroform only after the phenol/chloroform extraction? Did you get a clean interface between the phenol/chloroform and aqueous layer? If not, you should do another extraction.
phage434 on Mon Apr 4 20:31:57 2011 said:
Did you extract with chloroform only after the phenol/chloroform extraction? Did you get a clean interface between the phenol/chloroform and aqueous layer? If not, you should do another extraction.
The protocol I used is the following:
--> phenol/chloroform extraction
--> chloroform extraction
--> incubation of the aqueous phase with RNase
--> precipitation with 100% ice-cold ethanol
--> ethanol removal and resolubilization of dry DNA precipitate with sterile water
The interface between the phenol/chloroform and aqueous layer wasn't clean enough. There was a thin white-colored phase that it was difficult to avoid when removing the aqueous phase.
When you say that I should do another extraction what do you mean? To repeat the extraction with chloroform alone?
Thank you very much!
No, repeat the phenol/chloroform extraction. A second chloroform only extraction wouldn't hurt either. You might want to look into phase lock gels (5Prime company) to help with aqueous phase separation.
phage434 on Tue Apr 5 01:44:16 2011 said:
No, repeat the phenol/chloroform extraction. A second chloroform only extraction wouldn't hurt either. You might want to look into phase lock gels (5Prime company) to help with aqueous phase separation.
Just to make sure I understood what you said... you mean to do another phenol/chloroform extraction immediately after the first one and then one or two chloroform-only extractions. I will try it! Would a second ethanol precipitation help?
Thank u!
It might help, but not as much as the other things. A second wash with 70% ethanol will help in removing excess salt, if that matters in your experiment.
you should do the second extraction after the rnase treatment to get rid of the protein.
The white phase between the aqueous phase and the phenol are proteins. Looks like you pipetted them and it would explain why your A260/280 isn't good.