Samples can be stored at this point at À20
C for up to 7 days.
8. Enrich DNA Fragments.
• Remove one tube each of PCR Master Mix and PCR Primer Cocktail from À20
C
and thaw at RT—centrifuge briefly.
• Remove the AMPure XP Beads from 4
C and warm to RT at least 30 min.
• Remove Resuspension Buffer and samples from À20
C and thaw to RT. Briefly
centrifuge samples at 280Âg for 1 min.
• Prepare new 0.5 ml PCR tubes.
• Aliquot the appropriate volume of each reagent (with 10% excess per tube) into strip
tubes. Cap tubes and keep on ice until needed. Remaining content can be re-store at
À20
C.
• Add 5μl of thawed PCR Primer Cocktail to each sample.
• Add 25μl of thawed PCR Master Mix to each sample. Pipette up and down 10Â to
mix thoroughly.
• Run PCR in the pre-heated thermal cycler with closed lid using the following profile:
1 cycle
98
C
30 s
Use heated lid at 100
C
15 cycles
98
C
1 0 s
60
C
3 0 s
72
C
3 0 s
1 cycle
72
C
5 min
4
C
Hold
• Vortex the pre-warmed AMPure Beads until they are completely dispersed.
• Add 50μl of the beads to each sample containing 50μl of PCR amplified library and
pipette up and down 10Â to mix thoroughly.
• Incubate the PCR tubes at RT 15 min.
• Transfer samples to the magnetic stand at RT for at least 5 min making sure the liquid
clears.
• Remove and discard 95μl of the supernatant from each sample without disturbing the
beads. Some liquid may remain in the wells. Remember to change tips.
• Leave samples on the magnetic stand while performing the 80% ETOH washing steps.
• Add 200μl of freshly prepared 80% EtOH to each sample without disturbing the beads.
• Incubate the samples at RT at least 30 s, then remove and discard all the supernatant
from each sample without disturbing the beads. Remember to change tips.
• Repeat ETOH wash.
Appendix
199