DNA purification - (Oct/06/2005 )
I get something very odd after purifying my DNA with ARNase and a mixture of pancreatic proteases. phenol/chloroform extraction and then ethanol precipitation then resuspending in a TE buffer
My 260nm reading for my not purified DNA was 0.616 which gave me 30.8ug and then big surprise my 260nm reading for the purified DNA was 0.702 and it gives me 35.1ug.
I don't understand the logic behind this, it should be the opposite since there was RNA in the not purifed sample and it also absorbs at 260, there's no way that I have more DNA after purifying than before.
Now for the ratio 260/280, the not purified sample gives me 1.940 which makes sense, yet my 260/280 for the pure is 2.60 rather than the predicted 1.80.
For the non pure DNA, the 280 reading was 0.317 while for the pure DNA it was 0.270, this is the only piece of info which makes sense, phenol and aromatic amino acids absorb light at 280, so with the proteases we eliminated some of the proteins thus reducing the absorbance.
The experiment was done in a biochemisty class and all the other teams had similar results so I know that I didnt do anything wrong in protocol, unless we all messed up in the same fashion.
Any help would be greatly appreciated. I really dont see what can contaminate the DNA sample so badly.
Thanks in advance
Did you make sure you completely removed all of the ethanol, and allowed the pellet to dry? A common cause of a 260:280 above 2 is ethanol contamination.