• Remove samples from the thermal cycler.
• Add 5μl Stop Ligase Buffer [13] to each sample and mix by gently pipetting up and
down 10Â.
• Vortex the pre-warmed AMPure XP Beads until fully dispersed.
• Add 42μl of beads to each sample. Gently pipette up and down 10Â to mix well.
• Incubate samples at RT 15 min.
• Place samples on the magnetic stand at RT for at least 5 min making sure liquid clears.
• Remove and discard 79.5μl of supernatant from each sample. DO NOT DISTURB
BEADS. Change tips after each removal.
• Leave samples on the magnetic stand while performing the 80% ETOH washing steps.
• Add 200μl freshly prepared 80% EtOH to each sample without disturbing the beads.
• Incubate samples at RT for at least 30 s. Then remove and discard all the supernatant
from each sample without disturbing the beads. Change tips between wells.
• Repeat ETOH wash.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Remove samples from magnetic stand and resuspend the dry pellet in each sample with
52.5μl of Resuspension Buffer. Gently pipette up and down 10Âs to mix.
• Incubate samples at RT for 2 min.
• Place samples on the magnetic stand and incubate at least 5 min—make sure liquid
clears.
• Transfer 50μl of clear supernatant from each sample to the corresponding new 0.5 ml
tubes. Some residual liquid may remain in each sample.
• Vortex the AMPure XP beads to disperse and add 50μl of beads into each sample for a
second clean-up. Pipette up and down 10Â to mix thoroughly.
• Incubate samples at RT for 15 min.
• Place the samples on the magnetic stand at RT for 5 min making sure the liquid has
cleared.
• Remove and discard 95μl of the supernatant from each sample without disturbing the
beads. Some liquid may remain in each sample. Remember to change tips between
wells.
• Leave samples on the magnetic stand while performing the 80% ETOH washing steps.
• Add 200μl freshly prepared 80% EtOH to each sample without disturbing the beads.
• Incubate samples at RT for at least 30 s, then remove and discard all of the supernatant
from each sample without disturbing beads. Change tips after each removal.
• Repeat ETOH wash.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Remove samples from magnetic stand and resuspend the dry pellet in each sample with
22.5μl of Resuspension Buffer. Gently pipette up and down 10Â to mix.
• Incubate samples at RT 2 min.
• Place samples on the magnetic stand at RT for at least 5 min making sure liquid clears.
• Transfer 20μl of clear supernatant from each sample to the corresponding new 0.5 ml
PCR tubes (PCR). Some residual liquid may remain in each sample.
198
Appendix
• Add 5μl Stop Ligase Buffer [13] to each sample and mix by gently pipetting up and
down 10Â.
• Vortex the pre-warmed AMPure XP Beads until fully dispersed.
• Add 42μl of beads to each sample. Gently pipette up and down 10Â to mix well.
• Incubate samples at RT 15 min.
• Place samples on the magnetic stand at RT for at least 5 min making sure liquid clears.
• Remove and discard 79.5μl of supernatant from each sample. DO NOT DISTURB
BEADS. Change tips after each removal.
• Leave samples on the magnetic stand while performing the 80% ETOH washing steps.
• Add 200μl freshly prepared 80% EtOH to each sample without disturbing the beads.
• Incubate samples at RT for at least 30 s. Then remove and discard all the supernatant
from each sample without disturbing the beads. Change tips between wells.
• Repeat ETOH wash.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Remove samples from magnetic stand and resuspend the dry pellet in each sample with
52.5μl of Resuspension Buffer. Gently pipette up and down 10Âs to mix.
• Incubate samples at RT for 2 min.
• Place samples on the magnetic stand and incubate at least 5 min—make sure liquid
clears.
• Transfer 50μl of clear supernatant from each sample to the corresponding new 0.5 ml
tubes. Some residual liquid may remain in each sample.
• Vortex the AMPure XP beads to disperse and add 50μl of beads into each sample for a
second clean-up. Pipette up and down 10Â to mix thoroughly.
• Incubate samples at RT for 15 min.
• Place the samples on the magnetic stand at RT for 5 min making sure the liquid has
cleared.
• Remove and discard 95μl of the supernatant from each sample without disturbing the
beads. Some liquid may remain in each sample. Remember to change tips between
wells.
• Leave samples on the magnetic stand while performing the 80% ETOH washing steps.
• Add 200μl freshly prepared 80% EtOH to each sample without disturbing the beads.
• Incubate samples at RT for at least 30 s, then remove and discard all of the supernatant
from each sample without disturbing beads. Change tips after each removal.
• Repeat ETOH wash.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Remove samples from magnetic stand and resuspend the dry pellet in each sample with
22.5μl of Resuspension Buffer. Gently pipette up and down 10Â to mix.
• Incubate samples at RT 2 min.
• Place samples on the magnetic stand at RT for at least 5 min making sure liquid clears.
• Transfer 20μl of clear supernatant from each sample to the corresponding new 0.5 ml
PCR tubes (PCR). Some residual liquid may remain in each sample.
198
Appendix