3. Purify using KAPA pure bead purification as in steps 4–15.
4. Equilibrate beads at room temperature and resuspend beads.
5. Add 1.2Â volumes (60 μL in this case) of beads to each sample.
Mix by pipetting.
6. Incubate at room temperature for 15 min.
7. Place tubes on a magnetic stand to capture beads until the
liquid is clear (~5 min).
8. Carefully remove and discard supernatant.
9. Keep the tubes on the magnetic stand and wash the beads by
adding 200 μL of 80% ethanol and incubating for 30 s.
10. Carefully remove and discard the ethanol.
11. Repeat washing step for a total of two times.
12. Use a P200 to pipet off any remaining supernatant. Leave
10–15 min on stand with cap open to allow evaporation of
residual ethanol.
13. Add 50 μL of water and incubate for 5 min at room temperature to resuspend the beads.
14. Place the tubes in the magnetic stand to capture the beads until
liquid is clear.
15. Transfer the supernatant to a clean 1.5 mL tube.
16. Concentrate in a vacuum centrifuge to 15.1 μL.
17. Use 1 μL to determine the concentration by Nanodrop.
3.5 PCR to Amplify
Correctly Adapted
Molecules
1. Perform one reaction per library and a no-template control (see
Note 8).
2. Combine the following for each reaction (can be prepared as a
master mix) (50 μL final volume per reaction):
(a) 10 μL of 5Â Q5 buffer.
(b) 2.5 μL of 10 μM primer SSS401.
(c) 2.5 μL of 10 μM primer SSS402.
(d) 20 μL of 5 M betaine (see Note 6).
(e) 0.5 μL of Q5 polymerase.
(f) 0.4 μL of dNTP mix (mix is 25 mM each dNTP).
(g) 14.1 μL of concentrated cDNA produce (from step 16 in
Subheading 3.4) or molecular-grade water for
no-template control (note: if template concentrations differ, normalize all to the concentration of the most dilute
library).
3. Perform PCR as follows, with reaction volume set to 100 μL
(see Note 7):
(a) 98 ˚C for 3 min.
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4. Equilibrate beads at room temperature and resuspend beads.
5. Add 1.2Â volumes (60 μL in this case) of beads to each sample.
Mix by pipetting.
6. Incubate at room temperature for 15 min.
7. Place tubes on a magnetic stand to capture beads until the
liquid is clear (~5 min).
8. Carefully remove and discard supernatant.
9. Keep the tubes on the magnetic stand and wash the beads by
adding 200 μL of 80% ethanol and incubating for 30 s.
10. Carefully remove and discard the ethanol.
11. Repeat washing step for a total of two times.
12. Use a P200 to pipet off any remaining supernatant. Leave
10–15 min on stand with cap open to allow evaporation of
residual ethanol.
13. Add 50 μL of water and incubate for 5 min at room temperature to resuspend the beads.
14. Place the tubes in the magnetic stand to capture the beads until
liquid is clear.
15. Transfer the supernatant to a clean 1.5 mL tube.
16. Concentrate in a vacuum centrifuge to 15.1 μL.
17. Use 1 μL to determine the concentration by Nanodrop.
3.5 PCR to Amplify
Correctly Adapted
Molecules
1. Perform one reaction per library and a no-template control (see
Note 8).
2. Combine the following for each reaction (can be prepared as a
master mix) (50 μL final volume per reaction):
(a) 10 μL of 5Â Q5 buffer.
(b) 2.5 μL of 10 μM primer SSS401.
(c) 2.5 μL of 10 μM primer SSS402.
(d) 20 μL of 5 M betaine (see Note 6).
(e) 0.5 μL of Q5 polymerase.
(f) 0.4 μL of dNTP mix (mix is 25 mM each dNTP).
(g) 14.1 μL of concentrated cDNA produce (from step 16 in
Subheading 3.4) or molecular-grade water for
no-template control (note: if template concentrations differ, normalize all to the concentration of the most dilute
library).
3. Perform PCR as follows, with reaction volume set to 100 μL
(see Note 7):
(a) 98 ˚C for 3 min.
522
M. Carla Martini et al.