7 replies to this topic

[olga_m]

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!  

[phage434]

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.

[olga_m]

phage434, on 04 April 2011 - 12:31 PM, 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!

[phage434]

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.

[olga_m]

phage434, on 04 April 2011 - 05:44 PM, 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!

[phage434]

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.

[mdfenko]

you should do the second extraction after the rnase treatment to get rid of the protein.

[Maddie]

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.