• Add 12.5μl of thawed A-Tailing Mix [11] to each sample. Pipette up and down 10Â
to mix.
• Incubate samples in the pre-heated thermal cycler with closed lid at 37
C for 30 min
using the following profile:
37
C
30 min
Use heated lid at 100
C
70
C
5 min
4
C
Hold
• Immediately remove the samples from the thermal cycler—Immediately proceed with
Adapter Ligation!
7. Ligate Adapters
• Remove the appropriate RNA Adapter Index tubes (AR001-AR012, depending on
the RNA Adapter Indexes being used) and one tube of Stop Ligase Buffer
[13] from À20
C and thaw at RT.
• Remove Resuspension Buffer from 4
C and warm to RT.
• Remove the AMPure XP Beads from 4
C storage and warm to RT at least 30 min.
• Initiate thermal cycler profile: pre-heat to 30
C.
• Prepare new 0.5 ml PCR tubes (2Â, for Adapter ligation and PCR).
• Briefly centrifuge the thawed RNA Adapter Index tubes and Stop Ligase Mix.
• Add 2.5μl Resuspension Buffer [10] to each sample.
• Remove the DNA Ligase Mix [12] from À20
C immediately before use and leave in
À20
C benchtop storage cooler.
• Add 2.5μl of DNA Ligase Mix [12] directly from the À20
C benchtop storage cooler
to each sample.
• Return À20
C benchtop storage cooler back to freezer immediately after use.
• Add 2.5μl of the appropriate thawed RNA Adapter Index (AR001-AR012) to each
sample.
• Adjust the pipette to 40μl and gently pipette the entire volume up and down 10Â to
mix thoroughly.
• Change gloves after pipetting each adapter and clean pipette.
• Immediately store adapters at À20
C (sign as used).
• Incubate samples in the pre-heated thermal cycler with closed lid at 30
C for 10 min
using the following profile:
30
C
10 min
Use heated lid at 100
C
30
C
Hold
Appendix
197
to mix.
• Incubate samples in the pre-heated thermal cycler with closed lid at 37
C for 30 min
using the following profile:
37
C
30 min
Use heated lid at 100
C
70
C
5 min
4
C
Hold
• Immediately remove the samples from the thermal cycler—Immediately proceed with
Adapter Ligation!
7. Ligate Adapters
• Remove the appropriate RNA Adapter Index tubes (AR001-AR012, depending on
the RNA Adapter Indexes being used) and one tube of Stop Ligase Buffer
[13] from À20
C and thaw at RT.
• Remove Resuspension Buffer from 4
C and warm to RT.
• Remove the AMPure XP Beads from 4
C storage and warm to RT at least 30 min.
• Initiate thermal cycler profile: pre-heat to 30
C.
• Prepare new 0.5 ml PCR tubes (2Â, for Adapter ligation and PCR).
• Briefly centrifuge the thawed RNA Adapter Index tubes and Stop Ligase Mix.
• Add 2.5μl Resuspension Buffer [10] to each sample.
• Remove the DNA Ligase Mix [12] from À20
C immediately before use and leave in
À20
C benchtop storage cooler.
• Add 2.5μl of DNA Ligase Mix [12] directly from the À20
C benchtop storage cooler
to each sample.
• Return À20
C benchtop storage cooler back to freezer immediately after use.
• Add 2.5μl of the appropriate thawed RNA Adapter Index (AR001-AR012) to each
sample.
• Adjust the pipette to 40μl and gently pipette the entire volume up and down 10Â to
mix thoroughly.
• Change gloves after pipetting each adapter and clean pipette.
• Immediately store adapters at À20
C (sign as used).
• Incubate samples in the pre-heated thermal cycler with closed lid at 30
C for 10 min
using the following profile:
30
C
10 min
Use heated lid at 100
C
30
C
Hold
Appendix
197