20μl sized DNA sample
+ 30μl master mix
50μl.
PCR-cycler:
98
C
3 0 s
12Â
98
C
1 0 s
60
C
3 0 s
72
C
3 0 s
72
C
5 min
4
C
1
12. Clean up with AMPure beads
• 50μl after ligation.
• +55μl AMPure XP magnetic beads.
• Gently pipet up and down 10Â.
• 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.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Transfer 50μl in new tube for second clean-up.
13. Clean up with AMPure beads.
• 50μl after ligation.
• +55μl AMPure XP magnetic beads.
• Gently pipet up and down 10Â.
• 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 16μl EB, pipette the entire volume up and down 10 times to mix
thoroughly.
• 2 min at room temperature.
• ↺ 13,000 rpm, 1 s.
Appendix
207
+ 30μl master mix
50μl.
PCR-cycler:
98
C
3 0 s
12Â
98
C
1 0 s
60
C
3 0 s
72
C
3 0 s
72
C
5 min
4
C
1
12. Clean up with AMPure beads
• 50μl after ligation.
• +55μl AMPure XP magnetic beads.
• Gently pipet up and down 10Â.
• 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.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Transfer 50μl in new tube for second clean-up.
13. Clean up with AMPure beads.
• 50μl after ligation.
• +55μl AMPure XP magnetic beads.
• Gently pipet up and down 10Â.
• 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 16μl EB, pipette the entire volume up and down 10 times to mix
thoroughly.
• 2 min at room temperature.
• ↺ 13,000 rpm, 1 s.
Appendix
207