Trouble with DNA purity (A260/A280) - (Mar/05/2013 )
I've been using Quiagen Endo Free Maxi Prep to prepare some DNA. At the last step when you resuspend the dried pellet I always add 500 ul TE no matter the size of the pellet. I understand that a low A260 means that there can be impurites like RNA or if the pellet wasn't completely dry that would have an effect too. When I spec the Maxi I noticed that the maxi's with the higher concentrations have lower A260 values. Is this normal?
Here are a list of the DNA concentration/A260 values of my recent maxis:
Why is it that the concentration decreases as A260 increases? Also, can I use this tecnically unpure DNA to grow up more maxi's or is it useless now since the purity is so low?
Also why is it when I dilute the second maxi listed in more TE buffer (what I resupended the pellet in) I can get a concentration of 800 and an A260 of 1.8?
Any help is welcome! THANKS
What do you use for measuring the concentrations? Are you sure the values are not above the machine limit?
You can try dilute it 3 or more times and measure again.
Edit: Oh, you did actually try to dilute and got normal ratio values. So I think this is a problem with too high absorbance out of the machine limits.
Dilute all your samples and measure the concentrations within "safe" values, then multiply by dilution factor to get the original concentration (if you don't want to keep your DNA rather more diluted, which is good idea at least for PCR).
i dont think there is any problem with your procedure as u going to follow the kit protocol, but make sure your dnt use too much bacteria. because when you use beyond the suggestion limit your extraction wnt be efficient, esply in your case the first step P1 (with RNAse) lysis matters with RNA contamination. next check in another machine, nanodrop would be more accurate.
and rna will increase the a260 (also, purified rna will give a a260/a280 of ~2).
the ratio can be reduced by the presence of peptides.