PCR-cycler:
Pre-heated lid 00
C
3 7
C
30 min
70
C
5 min
4
C
1
4. Adapter Ligation
• Preparation of adapters (RNA Adapter Indices: AR001–AR016, AR018–AR023,
AR025, AR027):
• (at the moment Adapters of the TruSeq RNA-Seq-Kit).
• Centrifuge the Stop Ligation Buffer and appropriate/desired thawed RNA Adapter
tubes to 600 Âg for 5 s.
• Immediately before use, remove the Ligation Mix tube from À20
C and to À20
C
immediately after use.
• 30μl sample.
• +2.5μl Resuspension Buffer[1].
• +2.5μl Ligation Mix[4].
• +2.5μl thawed RNA Adapter Index.
• 37.5μl.
• Gently pipette the entire volume up and down 10Â to mix thoroughly.
• Centrifuge PCR samples at 280 g for 1 min.
• ! ligate for 10 min at 30
C (thermal cycler use heated lid at 100
C?)
• +5μl Stop Ligation Buffer [6]
– 42.5μl.
• Gently pipette the entire volume (42,5μl) up and down 10 times.
– +7.5μl H 2 O
– 50μl.
• Gently pipette the entire volume (50μl) up and down 10 times.
5. Clean up with AMPure beads.
• 50μl after ligation.
• +55μl AMPure XP magnetic beads.
• Gently pipet up and down 10 x.
• 5 min at room temperature.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Discard supernatant.
• 2 Â 500μl of 70% ethanol for 30 s, wash magnetic beads, discard supernatant.
• Air dry the beads on magnetic stand at RT for 2–5 min or at 37
C in a heatblock.
• Elute with 50 μl H 2 O, pipette the entire volume up and down 10 times to mix
thoroughly.
• 2 min at room temperature.
• ↺ 13,000 rpm, 1 s.
202
Appendix