First of all, I am a student who's doing a project for the first time. For simplicity's sake, the current goal is to plot a Fluorescence/ DNA concentration curve using propidium iodide.
Since propidium iodide can detect DNA outside cells, it can be used to detect DNA inside a buffer solution. The protocol is as follows:
1. Perform 10x serial dilutions on DNA standard to obtain 0.1 ug/mL, 0.01ug/mL, 0.001ug/mL and 0,0001ug/mL solutions of DNA. (Only containing DNA standard, TE buffer and nuclease free water)
2. Obtain a blank (Nuclease free water)
3. Pipette DNA solutions into 96 well plate (50uL). Pipette 50uL working solution of propidium iodide (Dissolved in water) into 96 well plate.
4. Incubate for 2 minutes in the dark.
5. Run plate in Bio-Tek microplate reader and detect fluorescence for the recommended emission and excitation wavelengths.
After doing this however, my results do not show a strong linear relationship between DNA concentration and fluorescence. the R^2 value is 0.1991. Could someone please tell me what went wrong? Thank you.
Try adding 0.01% Triton X-100 or Tween-20 to your buffer. This is to block the surface of the plate and keep the dye from sticking to it.
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