(c) Final extension: 65
C, 5 min, 1 cycle.
(d) Hold at 4
C.
39. Vortex Agencourt AMPure XP Beads to resuspend and bring
to room temperature.
40. Add 45 μL (0.9Â) of resuspended Agencourt AMPure XP
Beads to the PCR reaction (~50 μL). Mix well on a vortex
mixer or by pipetting up and down at least ten times.
41. Incubate for 5 min at room temperature.
42. Quickly spin the tube in a microcentrifuge and place the tube
on an appropriate magnetic rack to separate beads from the
supernatant. After the solution is clear (about 5 min), carefully
remove and discard the supernatant. Be careful not to disturb
the beads that contain DNA targets.
43. Add 200 μL of freshly prepared 80% ethanol to the tube while
in the magnetic rack. Incubate at room temperature for 30 s,
then carefully remove, and discard the supernatant.
44. Repeat step 43 once for a total of two washing steps.
45. Air-dry the beads for 5 min while the tube is on the magnetic
rack with the lid open. Remove any residual ethanol after
5 min. Do not over dry the beads. This may result in
lower recovery of DNA target (see Note 9).
46. Remove the tube from the rack. Elute the DNA target from the
beads into 23 μL of 0.1Â TE. Mix well on a vortex mixer or by
pipetting up and down, quickly spin the tube in a microcentrifuge, and incubate for 2 min at room temperature. Place it in
the magnetic rack until the solution is clear.
47. Transfer 20 μL of the supernatant to a clean PCR tube and
store at À20
C.
3.3 Assess Library
Concentration
1. Before qPCR reaction setup, add 1 mL of Primer Premix (10Â)
to the 5 mL bottle of KAPA SYBR
® FAST qPCR Master Mix
(2Â) and mix by vortexing for 10 s. Record the date of Primer
Premix addition on the KAPA SYBR
® FAST qPCR Master Mix
bottle.
2. Perform each of the following 5 dilutions of the sample
libraries in 10 mM Tris–HCl, pH 8.0 + 0.05% Tween 20 (see
Note 15) to obtain samples diluted 1:32000 and 1:64000.
Dilutions
Serial dilutions
Sample
volume (μL)
Tris–HCl buffer
solution volume (μL)
Cumulative
dilution ratio
1 to 100
2 (from
neat sample)
198
100
1 to 40
5
195
4000
(continued)
RNAseq Workflow
495
C, 5 min, 1 cycle.
(d) Hold at 4
C.
39. Vortex Agencourt AMPure XP Beads to resuspend and bring
to room temperature.
40. Add 45 μL (0.9Â) of resuspended Agencourt AMPure XP
Beads to the PCR reaction (~50 μL). Mix well on a vortex
mixer or by pipetting up and down at least ten times.
41. Incubate for 5 min at room temperature.
42. Quickly spin the tube in a microcentrifuge and place the tube
on an appropriate magnetic rack to separate beads from the
supernatant. After the solution is clear (about 5 min), carefully
remove and discard the supernatant. Be careful not to disturb
the beads that contain DNA targets.
43. Add 200 μL of freshly prepared 80% ethanol to the tube while
in the magnetic rack. Incubate at room temperature for 30 s,
then carefully remove, and discard the supernatant.
44. Repeat step 43 once for a total of two washing steps.
45. Air-dry the beads for 5 min while the tube is on the magnetic
rack with the lid open. Remove any residual ethanol after
5 min. Do not over dry the beads. This may result in
lower recovery of DNA target (see Note 9).
46. Remove the tube from the rack. Elute the DNA target from the
beads into 23 μL of 0.1Â TE. Mix well on a vortex mixer or by
pipetting up and down, quickly spin the tube in a microcentrifuge, and incubate for 2 min at room temperature. Place it in
the magnetic rack until the solution is clear.
47. Transfer 20 μL of the supernatant to a clean PCR tube and
store at À20
C.
3.3 Assess Library
Concentration
1. Before qPCR reaction setup, add 1 mL of Primer Premix (10Â)
to the 5 mL bottle of KAPA SYBR
® FAST qPCR Master Mix
(2Â) and mix by vortexing for 10 s. Record the date of Primer
Premix addition on the KAPA SYBR
® FAST qPCR Master Mix
bottle.
2. Perform each of the following 5 dilutions of the sample
libraries in 10 mM Tris–HCl, pH 8.0 + 0.05% Tween 20 (see
Note 15) to obtain samples diluted 1:32000 and 1:64000.
Dilutions
Serial dilutions
Sample
volume (μL)
Tris–HCl buffer
solution volume (μL)
Cumulative
dilution ratio
1 to 100
2 (from
neat sample)
198
100
1 to 40
5
195
4000
(continued)
RNAseq Workflow
495