- Add 1μL of 10 mM dNTPs and 0.5μL of 0.5 pmol/μLRT#
primer to each sample (seeNote 20). - Denature RNA using the following thermocycler program:
(a) 70 C 5 min
(b) 25 C Forever- Add 13μL of freshly prepared reverse transcription mix to each
sample and mix by pipetting. Carry out reverse transcription
using the following settings:
(a) 25 C 5 min
(b) 42 C 20 min
(c) 50 C 40 min
(d) 80 C 5 min
(e) 4 C Forever- Add 1.65μL of 1 M NaOH to each sample and incubate at
98 C for 20 min in order to cause alkaline hydrolysis of RNA
that can interfere with the CircLigase reaction. - Neutralize samples by adding 20μL of HEPES pH 7.3.
Optional: At this stage samples to be multiplexed can be
combined to reduce the number of samples to work with. It
is recommended that negative controls are kept separate
from working samples. - Add 350μL TE buffer, 0.75μL GlycoBlue, and 40μL3
sodium acetate pH 5.5, and mix. Add 1 mL of ice-cold 100%
ethanol, mix again, then precipitate overnight at 20 C.
3.9 cDNA
Purification and Size
Selection
- Spin samples for 20 min at 4C and>18,000g.
- Remove supernatant leaving ~50μL around blue pellet. Add
1 mL of ice-cold 80% ethanol (do not re-suspend) and spin
samples for additional 10 min at 4C and>18,000g. - Carefully remove all supernatant. Use a P10 pipette tip for last
fewμL of supernatant around pellet. - Air-dry pellet at room temperature for 5 min with lid opened.
- Resuspend pellet in 12μLof1TBE-UREA loading buffer
prepared using nuclease free water. Also add 6μLof2TBE-
UREA loading buffer to 6μL of a 1:30 dilution of low molec-
ular weight marker. - Heat samples for 80C for 5 min directly before gel loading.
442 Christopher R. Sibley