Materials Required: Agarose, Buffers and reagents for horizontal gel as discussed before.

Procedure: The value of the relative migration (Rf) of each DNA band is calculated from the agarose gel. The values of relative migration (Rf) and size of the DNA is used to draw the calibration curve to calculate the size of the unknown DNA samples.

Lab Experiment 17.2 : Analyze the interaction between single stranded binding protein and different DNA species.

Background Information: DNA is a negatively charged molecule and it interact with positively charged protein to form DNA-protein complex. The size and the hydrodynamic volume changes when DNA is interacting with protein to form DNA-protein complex.

 

Hence, at the end of the experiment, we can be able to understand several aspects of DNA-protein interaction:

1. Whether protein-X has a affinity for DNA and the interaction is specific or non-specific in nature.

2. What will be affinity parameters of the interaction of DNA to protein in making DNA-protein complex?

Materials Required: Agarose, TAE Buffer, EtBr, Pure SSB, DNA

Procedure: To study the DNA-protein interaction, a fix amount of DNA is incubated with the increasing concentration of protein (Figure 17.4). Due to the formation of DNA-protein complex, the hydrodynamic volume of the complex increases and a shift in band is observed. The DNA has a extended structure and it provides docking site for several protein molecules such as single stranded binding protein (SSB).

Observation: As a result, a gradual shift in DNA band will be observed until the DNA binding site is not saturated with the protein molecules.