• Add 8.5μl Elute, Prime, Fragment High Mix [7] to each sample and gently pipette up
and down 10Â to mix thoroughly.
• Store Elute, Prime, Fragment High Mix at À20
C.
• Incubate samples in the thermal cycler using the following profile:
94
C
8 min
Use heated lid at 100
C
4
C
Hold
• Remove the samples when 4
C is reached and centrifuge briefly.
• Proceed immediately to First Strand Synthesis.
4. Synthesize First Strand cDNA
• Thaw one tube of First Strand Synthesis Act D Mix [8], spin briefly, and place on ice.
• Add 1μl Superscript II [8] to 9μl First Strand Synthesis D Mix (8μl per sample
needed; scale up volume as needed).
• Mix well by finger flicking—DO NOT VORTEX.
• Centrifuge briefly.
• Store First Strand Synthesis Act D Mix at À20
C immediately after use.
• Add 8μl of Super Script II supplemented First Strand Synthesis Act D Mix to each
sample.
• Gently mix by pipetting up and down 6Â.
• Centrifuge briefly.
• Incubate samples in a thermal cycler using the following profile:
25
C
10 min
Use heated lid at 100
C
42
C
15 min
70
C
15 min
4
C
Hold
• When the cycler reaches 4
C remove samples and proceed immediately to the Second
Strand Synthesis.
5. Synthesize Second Strand cDNA
• Thaw Second Strand Marking Master Mix [9] at RT and warm 4
C Resuspension
Buffer to RT.
• Warm AMPure XP Beads to RT
30 min; rotate beads at 6 rpm.
• Initiate thermal cycler: pre-heat to 16
C.
• Briefly Centrifuge Thawed Second Strand Marking Master Mix [9].
• Add 5μl Resuspension buffer [10] into each sample.
Appendix
195