DNA was extracted from urine and plasma samples using an optimized Methylation On Beads (MOB) protocol. MOB is a process that allows DNA extraction and bisulfite conversion in a single tube via the use of silica super Magnetic Beads (10 (link),17 (link)). This approach yields a 1.5- to 5-fold improvement in extraction efficiency compared with traditional techniques (10 (link),18 (link)). We optimized the MOB protocol which was previously a 24-hour protocol to a 6-hour protocol. We are newly describing MOB protocol for the isolation of DNA from the urine.
In the improved protocol, plasma samples were incubated with Proteinase K (10 mg/mL) (New England Biolabs Co.: P8107s) and Buffer AL (Qiagen, Co.: 19075) at 55°C for 1 hour. During the DNA bisulfite treatment procedure, CT lightning conversion reagent was added and incubated at 98°C for 10 mins and then at 70°C for 1 hour.
For DNA extraction from urine 150 μL of Proteinase K (10 mg/mL) (New England Biolabs Co.: P8107s) was added to 3mL of urine followed by 3mL of Buffer AL (Qiagen, Co.: 19075) and incubated in a water bath at 55 °C for 1 hour. After digestion 3ml of 100% of isopropanol and 150ul of Magnetic Beads (Promega, Co: magnesi KF-MD1471) were added to the sample to bind the DNA. Plasma and urine samples were prepared with parallel digestion workflows running concurrently (10 (link)).
In the improved protocol, plasma samples were incubated with Proteinase K (10 mg/mL) (New England Biolabs Co.: P8107s) and Buffer AL (Qiagen, Co.: 19075) at 55°C for 1 hour. During the DNA bisulfite treatment procedure, CT lightning conversion reagent was added and incubated at 98°C for 10 mins and then at 70°C for 1 hour.
For DNA extraction from urine 150 μL of Proteinase K (10 mg/mL) (New England Biolabs Co.: P8107s) was added to 3mL of urine followed by 3mL of Buffer AL (Qiagen, Co.: 19075) and incubated in a water bath at 55 °C for 1 hour. After digestion 3ml of 100% of isopropanol and 150ul of Magnetic Beads (Promega, Co: magnesi KF-MD1471) were added to the sample to bind the DNA. Plasma and urine samples were prepared with parallel digestion workflows running concurrently (10 (link)).
