Genomic DNA purity...???
Posted 04 April 2011 - 11:52 AM
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!
Posted 04 April 2011 - 12:31 PM
Posted 04 April 2011 - 12:46 PM
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!
Posted 04 April 2011 - 05:44 PM
Posted 04 April 2011 - 10:23 PM
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?
Posted 05 April 2011 - 03:42 AM
Posted 05 April 2011 - 12:26 PM
genius does what it must
i do what i get paid to do
Posted 09 April 2011 - 06:07 PM