68
C
5 min
Use heated lid at 100
C
• Remove samples from thermal cycler and incubate 1 min at RT.
• Vortex the warmed rRNA Removal Beads vigorously to completely resuspend the
beads.
• Add 35μl of the rRNA Removal Beads to new 0.5 ml PCR tubes.
• Transfer entire contents of each sample (20μl) into the new tubes containing rRNA
Removal Beads.
• Adjust pipette to 45μl, then with the tip of the pipette at the bottom of the tube, pipette
quickly up and down 20 times to mix thoroughly.
• It is important to pipette up and down quickly to ensure thorough mixing. Insufficient
mixing leads to lower levels of rRNA depletion.
• Prevent solution from foaming.
• Incubate the samples at RT for 1 min.
• Place the samples on the magnetic stand for 1 min. at RT.
• Transfer all supernatant from each sample into a new 0.5 ml PCR tube.
• Return rRNA Removal Beads [3] to 4
C.
• Place the samples on the magnetic stand for 1 min at RT. (Repeat as necessary until there
are no beads remaining.)
• Vortex the AMPure XP beads [5] until fully dispersed then add 99μl of AMPure XP
beads [5] to each sample containing ribosomal depleted RNA. Pipette up and down
gently 10Â to mix.
• Incubate samples at RT for 15 min.
• Store AMPure XP Beads at 4
C.
• Place the samples on the magnetic stand at RT for 5 min—make sure beads are
completely deposited on side of tubes.
• Remove and discard all of the supernatant from each sample.
• Leave samples on the magnetic stand and wash wells with 200μl freshly prepared 80%
ETOH [4]—DO NOT DISTURB BEADS.
• Incubate samples at RT˚˜ 30 s. Remove ETOH using pipette—DO NOT DISTURB
BEADS.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Briefly centrifuge thawed RT Elution Buffer [6] at 600Âg for 5 s.
• Add 11μl Elution Buffer [6] to each sample and gently pipette entire volume 10Âs to
mix thoroughly.
• Store Elution Buffer at 4
C.
• Incubate samples at RT for 2 min.
• Place samples on the magnetic stand for 5 min. at RT.
• Transfer 8.5μl of the supernatant into new 0.5 ml PCR tubes.
194
Appendix
C
5 min
Use heated lid at 100
C
• Remove samples from thermal cycler and incubate 1 min at RT.
• Vortex the warmed rRNA Removal Beads vigorously to completely resuspend the
beads.
• Add 35μl of the rRNA Removal Beads to new 0.5 ml PCR tubes.
• Transfer entire contents of each sample (20μl) into the new tubes containing rRNA
Removal Beads.
• Adjust pipette to 45μl, then with the tip of the pipette at the bottom of the tube, pipette
quickly up and down 20 times to mix thoroughly.
• It is important to pipette up and down quickly to ensure thorough mixing. Insufficient
mixing leads to lower levels of rRNA depletion.
• Prevent solution from foaming.
• Incubate the samples at RT for 1 min.
• Place the samples on the magnetic stand for 1 min. at RT.
• Transfer all supernatant from each sample into a new 0.5 ml PCR tube.
• Return rRNA Removal Beads [3] to 4
C.
• Place the samples on the magnetic stand for 1 min at RT. (Repeat as necessary until there
are no beads remaining.)
• Vortex the AMPure XP beads [5] until fully dispersed then add 99μl of AMPure XP
beads [5] to each sample containing ribosomal depleted RNA. Pipette up and down
gently 10Â to mix.
• Incubate samples at RT for 15 min.
• Store AMPure XP Beads at 4
C.
• Place the samples on the magnetic stand at RT for 5 min—make sure beads are
completely deposited on side of tubes.
• Remove and discard all of the supernatant from each sample.
• Leave samples on the magnetic stand and wash wells with 200μl freshly prepared 80%
ETOH [4]—DO NOT DISTURB BEADS.
• Incubate samples at RT˚˜ 30 s. Remove ETOH using pipette—DO NOT DISTURB
BEADS.
• Air dry samples on heatblock at 37
C 2–5 min. (check beads in-between.)
• Briefly centrifuge thawed RT Elution Buffer [6] at 600Âg for 5 s.
• Add 11μl Elution Buffer [6] to each sample and gently pipette entire volume 10Âs to
mix thoroughly.
• Store Elution Buffer at 4
C.
• Incubate samples at RT for 2 min.
• Place samples on the magnetic stand for 5 min. at RT.
• Transfer 8.5μl of the supernatant into new 0.5 ml PCR tubes.
194
Appendix