6. Run PCR in a thermocycler according to the following
program:
Temperature (
C)
Cycles
Time (s)
98
1
120
98
22
10
50
20
72
20
72
1
120
4
–
3.5 Clean-up
1. Pool 5μL from each PCR (from step 6 under Subheading 3.4)
into a 1-well culture block. This is the pooled sequencing
library.
2. Transfer a volume (equal to that of the pooled sequencing
library) of SPRI beads, stored at 4
C, to a microcentrifuge
tube or conical tube (see Note 11).
3. Equilibrate to RT for 30 min.
4. Set aside 100μL of the pooled sequencing library to run quality
control checks. Add the remaining volume of the library to an
equal volume of RT SPRI beads.
5. Pipette slowly up and down ten times, aspirating the suspension from the bottom of the tube and dispensing near the top
of the suspension. It is essential that the suspension is well
mixed.
6. Incubate at RT for 20 min.
7. Distribute the suspension across 1.5 mL microcentrifuge
tubes, with 500μL in each.
8. Put the tubes into a magnetic rack and wait 3 min for the beads
to be completely pulled out of suspension.
9. Remove supernatant in two aspirations in order not to disturb
the SPRI beads: first remove 300μL, then the final 200μL.
Leave some supernatant behind if there is a risk of aspirating
beads.
10. Add 900μL 80% (v/v) EtOH to each microcentrifuge tube,
wait 30 s, and remove and discard the supernatant.
11. Repeat this wash (step 9) twice more.
12. After removing the final supernatant, pipette out any drops of
ethanol at the bottom of tubes and leave the tubes to dry with
lids open for 15 min.
13. Remove the microcentrifuge tubes from the magnetic racks.
336
Eachan O. Johnson and Deborah T. Hung
program:
Temperature (
C)
Cycles
Time (s)
98
1
120
98
22
10
50
20
72
20
72
1
120
4
–
3.5 Clean-up
1. Pool 5μL from each PCR (from step 6 under Subheading 3.4)
into a 1-well culture block. This is the pooled sequencing
library.
2. Transfer a volume (equal to that of the pooled sequencing
library) of SPRI beads, stored at 4
C, to a microcentrifuge
tube or conical tube (see Note 11).
3. Equilibrate to RT for 30 min.
4. Set aside 100μL of the pooled sequencing library to run quality
control checks. Add the remaining volume of the library to an
equal volume of RT SPRI beads.
5. Pipette slowly up and down ten times, aspirating the suspension from the bottom of the tube and dispensing near the top
of the suspension. It is essential that the suspension is well
mixed.
6. Incubate at RT for 20 min.
7. Distribute the suspension across 1.5 mL microcentrifuge
tubes, with 500μL in each.
8. Put the tubes into a magnetic rack and wait 3 min for the beads
to be completely pulled out of suspension.
9. Remove supernatant in two aspirations in order not to disturb
the SPRI beads: first remove 300μL, then the final 200μL.
Leave some supernatant behind if there is a risk of aspirating
beads.
10. Add 900μL 80% (v/v) EtOH to each microcentrifuge tube,
wait 30 s, and remove and discard the supernatant.
11. Repeat this wash (step 9) twice more.
12. After removing the final supernatant, pipette out any drops of
ethanol at the bottom of tubes and leave the tubes to dry with
lids open for 15 min.
13. Remove the microcentrifuge tubes from the magnetic racks.
336
Eachan O. Johnson and Deborah T. Hung