Genomic DNA purity PROBLEM

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Olga_m
Olga_m's picture
Genomic DNA purity PROBLEM

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!

Ivan Delgado
Ivan Delgado's picture
Unfortunately when you have a

Unfortunately when you have a 260/280 ratio as low as 1.17 you are likely measuring something other than DNA. The most likely scenario here is that you do not have much DNA in your extraction. I would extract again, or at least run your DNA in a gel to see if there is anything there. 

Biju
Biju's picture
Hi Olga

Hi Olga
Are you sure that you did not loose the DNA in one of the transfer step in the protocol?If you are sure that you have not missed it during transfer ,I will check for
1. morphology of cells by microscopy
2. cell nos before extraction
3. Quality of the eqilibrated phenol(Are you using commercial equilibrated phenol or in-house made?)to begin with.
Biju Joseph

Rajeshwari patel
Rajeshwari patel's picture
Hi,

Hi,

I agree with IVAN,

and in gel IF you see any DNA you can gel purify it
 
Thanks
Rajeshwari