Incredibly high 260/280 ratio...

[zera]

Hello,
I'm trying to do 3C experiments and I have all sort of problems. One of them being that after purification (2 phenol/IAA and 1 chloroform extranctions + ethanol precipitation), when I try to measure my DNA concentration with a nanodrop, I have incredibly high 260/280 ratio (between 4 and 6!!!). I read all sort of documents explaining the different kind of contaminants that I could have, but none of them could explain these ratio : the spectrum look normal (no shift of any sort anywhere). I'm pretty sure that I don't have any ethanol left in my sample. BUt I could have DNA degradation maybe? (I read somewhere that nucleotides could increase the 260/280 ratio). ANyway, if anyone had aby suggestion, that would be a huge help! Thanks!

[relaxin]

Phenol/chloroform extraction often left lots of impurities that may give you wierd A260/A280 ratio. In the good days, we normally remove the impurities by dialysis againt TE buffer before ethanol precipitation.

[zera]

OK. I read somewhere that diluting the DNA before precipitation could help getting rid if the impureties, that's the same principle, isn't it? There's some DTT in the ligation buffer rigth before extraction/precipitation, could it interfere with OD 260? And what should I do now, would another round of ethanol precipitation help? Thank you for your help!

[mchlbrmn]

If you dilute it can help with impurities with limited solublility in the solvent, but impurities that coprecipitate easily in alcohol will continue to do so. Dialysis will remove anything below the cut off size.
I don't know what the problem is. You could try measuring the OD of the various components present to see what has a high absorbance at A260 that could be the problem. (I assume the OD was zeroed properly and washed after precipitation.)
(PS. I assume the IAA was in the CHCl3, not the phenol. (Not that this would cause the problem.))

[zera]

yes, in fact it is 2 phenol/chloroform/IAA extractions and one pure chloroform.
Yes again, the OD was zeroed properly, and yes I did a 70% ethanol wash after the precipitation.
Good idea, I could try to OD the ligation buffer and see what its spectrum looks like...
I will also try to quantify my DNA using something else : it looks like I have a big overestimation of the DNA amount (I try to do qPCR afterwards). But as I don't know what the problem is, I don't know if it's indeed that the amount is wrong, or if the impurities interfere with the following steps...
Anyway, thanks for your help !