Lab Experiment 19.1: Detect the LDH gene in the human genome using southern blotting.

Introduction: In southern blotting, the genomic DNA is digested with the EcoRI or BamHI and the DNA fragments are resolved on the agarose gel. The gel is incubated in an alkaline solution to denature the double stranded DNA to single stranded form. DNA is transferred on the nitrocellulose membrane by capillary action by applying a uniform pressure either by suction pressure or by placing wet paper towels. The membrane is incubated with a non-specific DNA such as sonicated calf thymus genomic DNA to block the binding sites on the membrane. A single stranded radioactive probe is added to the membrane and allowed to bind. Membrane is washed and the blot is developed by autoradiography. The DNA fragment complementary to the probe sequence binds the radioactive probe and give positive signal (Figure 19.1).

Figure 19.1: General Over-view of different steps in Southern Blotting.