Isolation of Circulating Cell-free DNA (cfDNA): A Silica-based Membrane Procedure to Extract cfDNA from Blood Plasma of Cancer Patients

To isolate circulating cell-free DNA from a patient plasma sample, add 100 microliters of Proteinase K and 800 microliters of Lysis buffer supplemented with one microgram of carrier RNA to 1 milliliter of patient plasma. Thoroughly pulse vortex the solution for 30 seconds before incubating for 30 minutes at 60 degrees Celsius. At the end of the incubation, add 1.8 milliliters of binding buffer to the tube and thoroughly mix with 15 to 30 seconds of pulse vortexing.

After a 5-minute incubation on ice, insert a silica membrane column into a vacuum apparatus connected to a vacuum pump and firmly insert a 20-milliliter tube extender into the open column to prevent sample leakage. At the end of the incubation, carefully pour the mixture into the tube extender and switch on the vacuum pump. When all of the lysate has completely run through the columns, switch off the vacuum pump, release the pressure to zero millibars, and carefully discard the tube extender without contaminating the adjacent columns.

Transfer the column into a collection tube for centrifugation to remove any residual lysate. After discarding the flow-through, add 600 microliters of Wash Buffer-1 to the column for a second centrifugation. After discarding the flow-through, centrifuge the column again with 750 microliters of Wash Buffer-2. After discarding the flow-through, add 750 microliters of 96% to 100% ethanol to the column for an additional centrifugation.

Transfer the column into a new 2-milliliter collection tube for another centrifugation before placing the membrane column assembly into a new 2-milliliter collection tube at 56 degrees Celsius for 10 minutes. At the end of the incubation, transfer the column into a new 1.5-milliliter elution tube and add 50 microliters of elution buffer to the column for a 3-minute incubation at room temperature. Then, centrifuge the recovered solution for 1 minute at 20,000 x g to elute the nucleic acids.