• Add 20μl of thawed Second Strand Marking Master Mix [9] to each sample. Gently
pipette up and down 6Â to mix thoroughly.
• Store Second Strand Marking Master Mix at À20
C.
• Incubate samples on a pre-heated thermal cycler with closed lid at 16
C 1 h using the
following profile:
16
C
1 h
Do not use heated lid (program: HEADER)
16
C
Hold
• Remove samples and place at RT to equilibrate.
• Vortex the RT AMPure XP beads until fully dispersed then add 90μl of beads to each
sample containing 50μl of ds cDNA. Pipette up and down gently 10Âs to mix.
• Incubate samples at RT 15 min.
• Place samples on the magnetic stand at RT for 5 min—make sure beads are completely
deposited on side of tubes.
• Remove and discard 135μl of the supernatant from each sample.
• Some liquid may remain in the tubes—DO NOT DISTURB BEADS.
• Leave samples on the magnetic stand and wash with 200μl freshly prepared 80%
EtOH—DO NOT DISTURB BEADS.
• Incubate samples at RT 30 s. Remove ETOH using pipette—DO NOT DISTURB
BEADS.
• Repeat 80% ETOH wash.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Add 17.5μl Resuspension Buffer [10] to each sample. Mix up and down 10Â to
completely resuspend beads.
• Incubate the samples at RT for 2 min.
• Place samples on the Magnetic Stand at RT˚˜ for 5 min.
• Transfer 15μl of the supernatant containing the ds cDNA to new 0.5 ml tubes.
• Some liquid may remain in the wells—DO NOT DISTURB BEADS.
Time until this point: ~7 h
Samples can be stored at this point at À20
C for up to 7 days.
6. Adenylate 3
0 Ends
• Remove one tube of A-Tailing Mix [11] from À20
C and thaw at RT.
• A-Tailing Mix aliquots (á 50μl) and label tubes. Store at À20
C.
• Remove samples from À20
C storage and thaw at RT. Briefly centrifuge samples at
280Âg for 1 min.
• Initiate thermal cycler profile: pre-heat to 37
C.
• Add 2.5μl Resuspension Buffer [10] to each sample.
196
Appendix
pipette up and down 6Â to mix thoroughly.
• Store Second Strand Marking Master Mix at À20
C.
• Incubate samples on a pre-heated thermal cycler with closed lid at 16
C 1 h using the
following profile:
16
C
1 h
Do not use heated lid (program: HEADER)
16
C
Hold
• Remove samples and place at RT to equilibrate.
• Vortex the RT AMPure XP beads until fully dispersed then add 90μl of beads to each
sample containing 50μl of ds cDNA. Pipette up and down gently 10Âs to mix.
• Incubate samples at RT 15 min.
• Place samples on the magnetic stand at RT for 5 min—make sure beads are completely
deposited on side of tubes.
• Remove and discard 135μl of the supernatant from each sample.
• Some liquid may remain in the tubes—DO NOT DISTURB BEADS.
• Leave samples on the magnetic stand and wash with 200μl freshly prepared 80%
EtOH—DO NOT DISTURB BEADS.
• Incubate samples at RT 30 s. Remove ETOH using pipette—DO NOT DISTURB
BEADS.
• Repeat 80% ETOH wash.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Add 17.5μl Resuspension Buffer [10] to each sample. Mix up and down 10Â to
completely resuspend beads.
• Incubate the samples at RT for 2 min.
• Place samples on the Magnetic Stand at RT˚˜ for 5 min.
• Transfer 15μl of the supernatant containing the ds cDNA to new 0.5 ml tubes.
• Some liquid may remain in the wells—DO NOT DISTURB BEADS.
Time until this point: ~7 h
Samples can be stored at this point at À20
C for up to 7 days.
6. Adenylate 3
0 Ends
• Remove one tube of A-Tailing Mix [11] from À20
C and thaw at RT.
• A-Tailing Mix aliquots (á 50μl) and label tubes. Store at À20
C.
• Remove samples from À20
C storage and thaw at RT. Briefly centrifuge samples at
280Âg for 1 min.
• Initiate thermal cycler profile: pre-heat to 37
C.
• Add 2.5μl Resuspension Buffer [10] to each sample.
196
Appendix