Saliva DNA Extraction

Saliva samples were collected from volunteers of 18-25 years of the Districts Abbottabad and Mansehra, Pakistan. Proper instructions were provided to the volunteers for vigorously rinsing mouth for one and a half minutes, and brushing teeth thereafter. Each subject was given 3-5/mL of 5% sucrose solution for rinsing mouth for two to three minutes and then spit into 15/mL sterile specimen tube, the swab thus obtained were stored at -20 °C till processing with our modified protocol of Ralser [7]. DNA was isolated by taking 1.00/mL of liquid salivain 1.5/mL eppendorf tube. 100 µL of lysis solution (2/mL lysis buffer + 10 µL of Proteinase K + 3 µL of marcaptoethanol) was added to saliva sample and was mixed well. Sample was then incubated at 56 °C for 1:30 min. After incubation 600 µL of Phenol:Chloroform (1:1) solution was added and was incubated at once for 5 min at room temperature. Sample was then centrifuged at 10,000 rpm for 10 min and supernatant was transferred into a fresh tube very carefully. Equal volume of isopropanol was added to the subjected supernatant and was incubated at 20 °C for 20 min. Sample was centrifuged again at 10,000 rpm for 15 min and upper layer was discarded and pellet was washed with 70% ethanol. Ethanol was discarded after centrifugation at 8,000 rpm for 5 min and pellet was air-dried. Thirty micro litre of double distilled water was added to the dried DNA pellet and was incubated at 56 °C for 10 min. DNA quality and quantity was then measured with Agarose gel electrophoresis.