UV absorbance titration to determine binding stoichiometry

I previously posted a question but I think I did not explain correctly. I want to construct a Job plot using UV-Vis titration to find binding stoichiometry. I have 20uM of ligand solution and 20uM of DNA solution. Volume of each solution is 800uL. The total concentration of ligand and dsDNA remained unchanged(20uM) during entire titration. Ligand intercalates into dsDNA.


I want to know the protocol to do UV-Vis titration. Like how I should proceed. Do I need to prepare solution of different concentration everytime (for example ligand solution --- 20uM, take UV absorbance,
Now make a complex solution of say 18uM ligand and 2uM dsDNA and so on)


I have no idea what to do.


Thank you.


[![enter image description here](https://i.stack.imgur.com/wyOox.jpg)](https://i.stack.imgur.com/wyOox.jpg)




I can advice only in general methodic, I cannot say what you can afford wrt the particular solutions, regarding their stability and interaction.


If the volume of available solutions **is not** the limiting factor, the easiest way is to prepare extra solution mixture for each measurement.


* Take $x\ \pu{ \mu L}$ of the ligand solution
* Take $a - x\ \pu{\mu L}$ of DNA solution
* It gives $\frac{x}{a-x}$ ligand/DNA ratio.


Then you can measure the series of plot point absorbance vs ligand/DNA ratio.


If the volume of available solutions **is** the limiting factor, you may try the series of incremental additions of the ligand and following measurements.


You would take


* $V\_1\ \pu{ \mu L}$ of solution of the prior ratio $r\_1$
* add $V\_2\ \pu{ \mu L}$ of the ligand solution to get the new ratio:


$$r\_2 = \frac{r\_1(V\_1+V\_2) + V\_2}{V\_1}$$


But there are 2 cons:


* Higher cummulative experimental errors with excessive solution manupulations.
* Possible effects of stability and interaction of incrementally mixed solutions.


When you have measurement done, you can visially or numerically approximate the poins by 2 lines and to take the intersection as the point of equivalence.


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