Master mix for blunting DNA
1 x [μl]
x [μl]
DNA
Resuspension Buffer [1]
10
End Repair Mix [2]
40
100.00
• 50μl sample
• +50μl master mix
• 100μl
! 30 min @ 30
C in thermocycler
2. Clean up with AMPure beads
• Vortex the AMPure XP Beads [5] until they are fully dispersed.
• 100μl after polish DNA ends.
• +160μl 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 127.5μl supernatant.
• Again discard 127.5μl supernatant (not once 255μl because of the suck).
• 2 Â 500μl of 70% ethanol for 30 s, wash magnetic beads, discard supernatant.
• Air dry the beads on magnetic stand at RT C for 2–5 min or at 37
C in a heatblock.
• Elute with 17.5μl Resuspension Buffer, 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.
• Transfer 15μl in new tube.
3. Perform 3
0 -dA addition
Master mix for A-tailing DNA
1 x [μl]
x [μl]
Resuspension Buffer[1]
2.50
A-Tailing Mix[3]
12,5
15
15μl sample
+ 15μl master mix
30μl
Appendix
201