I am performing B to A transition of DNA in presence of ethanol and certain ligands. The working concentration of the sonicated DNA sample is 50ug/ml and it gives a smear centered around 600bp. To plot the transition curve I need to convert the CD (millidegrees) to molar circular dichroism by the formula CD(mdeg)/l(in mm) x conc (Molar nucleotide) x 3298. I have two sub questions:
1) Since the sonicated DNA is not exactly 600bp, how do I calculate the concentration in molar nucleotide?
2) If I do not know the exact MW (say for highly polymerized calf thymus DNA purchased from sigma which I eventually sonicated) how would I perform this conversion?
1 M (nucleotide) ~ 330 g/l of your DNA sample, according to the definition of molar concentration and taking into account that the MW of a nucleotide ~330 g/mole. Thus, you need not the MW of your DNA samle. 50 mg/ml of any DNA corresponds to 0.15 mM (nucleotide)
The DNA concentration per nucleotide can be determined by absorption spectroscopy using the molar absorption coefficient at 260nm as 6600 M-1cm-1.
Reichmann, M. E.; Rice, S. A.; Thomas, C. A.; Doty, P. J. Am. Chem.
Soc. 1954, 76, 3047-3053
@Polyanichko: You mean 50ug/ml (not 50mg/ml) DNA solution corresponds to 0.15mM nucleotide right? But if I calculate concentration by the formula
mM = (wgt of DNA/MW) x (1/vol in lit)
where MW=no of nts x wgt of one nt = 1200 x 330 (in my case)
then the conc of 50ug/ml DNA would be 0.126uM...
I am confused!!!
One could simply measure the absorbance of the DNA and use the molar absorption coefficient (6600 M-1cm-1) at 260nm to derive the exact molar concentration of the DNA.
Molar Concentration = Abs / molar absorption coefficient (considering you use 1cm path length cuvette).
This way you don't have to worry about the calculations. However, you can always cross-check.
I am trying to figure this out also. Has anybody know a full absorption coefficient 250-400nm spectra for DNA? Also what would the concentration of DNA be in a diploid genome 5microns radius?
@Patrick
Do you mean 1. molar absorption coefficient or 2. absorption spectra of DNA?
1. Molar absorption coefficient of DNA is 6600 M-1cm-1 at 260nm wavelength.
2. Absorption spectra of DNA will typically produce a Gaussian (bell-shaped) profile from 200nm to 310nm with a maximum peak absorbing at 260nm (assuming your sample is dilute enough or your instrument is capable of measuring highly concentrated samples)
For obtaining concentration of DNA in diploid genome (per nucleotides) you would need to know the organism from where it comes as you need to know the number of base-pairs.
I mean absorption spectra for DNA I found a spectra but it is only for an oligonucletide. Can I use this spectra as a surrogate as it is giving same 6600 M^-1cm^-1 at 260nm. The shape tails off from 325nm to 400nm. not exactly Gaussian shaped.
well in human skin so there are 6.6 x 10^9bp. is concentration = (6.6x10^9 x 2)/avg. no.)/volume of diploid nucleus?
thanks
P
You are right, the shape only resembles Gaussian/bell and is not exactly (was just to give an example). It will change depending on how pure the DNA sample is.
The extinction coefficient that I mentioned previously is for Calf-thymus DNA. But you could use it to get rough estimation of the DNA from genome. In any case, you will need a much diluted sample of your DNA, and after measuring the absorbance, use Beer-Lambert Law to determine the concentration using Extinction (absorption) coefficient as 6600 M^-1cm^-1. For more accurate estimation of concentration, you will need to find out the absorption coefficient of your diploid DNA (from the sequence). You can use some of the online tools to do that: http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
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