Tissue digestion

Tissue pellets obtained from the previous step, i.e. with or without ethanol rehydration, was digested with 500 µL lysis buffer (pH 7.8) and 5 µL proteinase K (200 µg/mL) incubated at 56°C in a shaker incubator for 6 h. The composition of the lysis buffer was 0.5 mol/L Tris HCl, 10 mmol/L EDTA, 100 mmol/L NaCl and 2% SDS. The tubes were agitated continuously at 25 g to ensure proper digestion. The following effects were examined by modifying the protocol as per our needs: effect of incubation time and effect of inactivating proteinase K.

To examine the effects of incubation time, the tubes with tissue pellet, lysis buffer, and proteinase K were incubated for different durations of time. The digestion was carried out for 6h, 12 h, overnight (24 h), 48 h, and 72 h. Tissues of similar sizes were used and the tubes were agitated all along and the temperature was maintained at 56°C (optimum temperature for proteinase K).

The function of proteinase K is to digest proteins and any harmful enzymes such as nucleases. But proteinase K itself being an enzyme may or may not hamper the quality and quantity of the extracted DNA. There is no evidence of proteinase K harming the quality or the quantity of the DNA. So in order to test this, we inactivated proteinase K after the complete incubation period in some of our samples. After the respective incubation period, the tubes were kept at 90°C in a block heater for about 20 min.