DNA quantitation - (Nov/18/2005 )
I am trying to quantitate genomic DNA from yeast by absorbance method according to which a DNA solution of concentration 50ug/ml will have an absorbance of 1 at 260nm through a 1 cm pathlength. So what I did was:
1. Take 2 ml of TE buffer in a cuvette. Set zero at 260nm.
2. Add 2 ul of DNA solution (dissolved in dw) to it.
3. Take absorbance at 260nm.
I got an absorbance of 0.058 which means I have 2.5 ug/ml of DNA. The extraction was not through standard kit, I just used lysis buffer followed by phenol extraction and then alcohol precipitation. I would like to know whether the procedure I followed for DNA quantitation was correct or not which may have led to the exaggerated result.
Moreover I am confused about the absorbance value. I have read that for a reliable A value it should be between 0.1 and 1.0 But in my case the value is always around 0.02 and 0.05.
Thanking in advance
The calculation goes like this for DNA: reading OD260x50xdilution factor= concentration in ug/ml. According to what you wrote I would calculate your DNA to be 2.9 mg/ml
If you want your reading to be in a higher range depending on the spec. you are using, just make a more concentrated DNA solution/
I also think that you should prepare a more concentrated sample. Usually I will dilute my sample to 40-50X, because I heard that the spectrometer will not perform well if the sample is too much diluted.