A Practical Guide to Cancer Systems Biology

16 A Practical Guide to Cancer Systems Biology

• Prepare FSM and SuperScript II mix at the ratio of 1μL SuperScript II
  for each 9μL FSM. Gently mix thoroughly and centrifuge briefly.
  Note: Return the FSM and SuperScript II to−15 to− 25 ◦C storage immediately after
  use.


• Add 8μL of FSM and SuperScript II mix to each sample. Gently pipette
  the entire volume up and down 10 times to mix thoroughly.


• Place the sample on the thermal cycler to synthesize the 1ststrand cDNA
  using the following program:
  25 ◦C for 10 minutes
  42 ◦C for 50 minutes
  70 ◦C for 15 minutes
  Hold at 4◦C


• Remove the sample from the thermal cycler, centrifuge briefly, and proceed
  immediately to 2ndstrand cDNA synthesis.

IV.RT-PCR: 2ndstrand synthesis

• Pre-heat the thermal cycler to 16◦C.


• Add 25μL of SSM to each sample. Gently pipette the entire volume up
  and down 10 times to mix thoroughly.


• Place the sample on the thermal cycler to synthesize the 2ndstrand cDNA.

16 ◦C for 60 minutes

• Vortex the AMPure XP beads until they are well dispersed, then add
  90 μL of well-mixed AMPure XP beads to each sample containing 50μL
  of double stranded cDNA. Gently pipette the entire volume up and down
  10 times to mix thoroughly.


• Incubate the sample at room temperature for 15 minutes.


• Centrifuge briefly and place the sample on magnetic stand at room
  temperature for 5 minutes to make sure that all beads are bound to the
  side of the wells.


• Remove and discard 135μL of supernatant from each sample.

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 200μLof
  freshly prepared 80% EtOH to each sample without disturbing the
  beads.