20 A Practical Guide to Cancer Systems Biology
Note: Leave the sample tubes on the magnetic stand while performing all the following
80% EtOH wash steps.- With the sample remaining on the magnetic stand, add 200μLoffreshly
prepared 80% EtOH to each sample without disturbing the beads. - Incubate the sample at room temperature for 30 seconds, then remove
and discard all of the supernatant from each well. - Repeat the 80% EtOH wash steps twice.
- Open the lid and let the sample tube stand at room temperature for
15 minutes to dry and then remove the sample from the magnetic stand. - Add 22.5μL RSB to each sample. Gently pipette the entire volume up
and down 10 times to mix thoroughly. - Incubate the sample at room temperature for 2 minutes.
- Centrifuge briefly and place the sample on the magnetic stand at room
temperature for 5 minutes. - Transfer 20μL of the clear supernatant to a new PCR tube.
Note: The purified DNA can be stored at−15 –− 25 ◦Cforupto7days.- (Optional) Transfer 1μL of the clear supernatant to a new PCR tube for
library validation.
VIII.PCR amplification
- Add 5μL of thawed PPC to each sample. Gently pipette the entire volume
up and down 10 times to mix thoroughly. - Add 25μL of thawed PMM to each sample. Gently pipette the entire
volume up and down 10 times to mix thoroughly. - Place the sample on the thermal cycler to amplify the adapter ligated
DNA fragments using the following program:
98 ◦C for 30 seconds
13 Cycles of:
− 98 ◦C for 10 seconds
− 60 ◦C for 30 seconds
− 72 ◦C for 30 seconds
72 ◦Cfor5minutes
Hold at 10◦C- Vortex the AMPure XP beads until they are well dispersed, then add
50 μL of well-mixed AMPure XP beads to each sample containing 50μL