Analyzing with Origin

you can use Origin online via https://weblogin.tudelft.nl/

Note: For a better look at the pictures, just click on the picture.

With origin we can finally analyze the time the HJ is in isoI and isoII. First of all, drag a .dat file that is created in to the top left corner of the table, then release it.

Now Origin will open the dat file, and you will see two columns.

- Right click on the header of the first column à Set as à Y.

- Select all the data in the table with Ctrl + A. C

- Go to “plot” in the menu bar à statistics à histogram

- It should look like this

- Left click on a bar of the histogram and select “Plot details”

- Go to the 3^rd tab called: “Data”

- Deselect automatic binning

- Now, make the bin size to 0.2 and begin to 0.

- Now click apply en check whether the histogram still looks like an exponential function.

- If it doesn’t, increase the bin size.

- Click on any bar and select “go to bin worksheet”

- It should now look like this

- Click on the column of counts

- Go to “plot” in the menu bar à columns/bars à column

- This will create a new histogram, which should look like the previous histogram

- It now should look like this

- Go to menu bar à Analysis à fitting à fit exponential (when possible: à open dialog)

- You should see this pop up

- Click on the button in the red circle (fit until converged)

- Ok

- Switch to report sheet

- Now a new table will open, displaying the graph propertjes

You need to repeat these steps for all .dat files (up and down)

A second analysis is the FRET histogram, drag the data of selected.dat into Origin. The first column is time, the second the donor (D), and the third the acceptor (A).

You need a fourth column for the FRET signal (=A/(D+A)). To set the formula, select the column, right click, Set Column Values, and apply the right formula (Col(C)/(Col(B)+Col(C))).

In a similar way as described above, make a histogram of all FRET values. You should get a graph with two peaks. Set the bin size to 0.02.

Right click on the graph, go to bin worksheet. From the GraphsBin select the first two columns, then go to Analysis, peaks&basesline, fit multiple peaks.

Double click on the two peaks, and two gaussians should be fitted. The peak height also tells you something about the dwelltimes/ type of Holliday junction.