Reverse Transcription 3-3
Two-Step RT-PCR
RT Reaction Mix
RT Reaction Mix
Component
Volume/Tube
(µL) Final Concentration
RNase-free water See below
a
a. The volume of RNase-free water (µL) will be 3.85–RNA sample volume in a 10-µL
reaction.
—
10X TaqMan RT Buffer 1.0 1X
25 mM MgCl
2
2.2 5.5 mM
deoxyNTPs Mixture (2.5 mM) 2.0 500 µM
per dNTP
Random Hexamers
b
(50 µM)
b. Random hexamers, oligo d(T)
16
, or sequence-specific reverse primers can be used for
primers of cDNA synthesis.
0.5 2.5 µM
RNase Inhibitor (20 U/L) 0.2 0.4 U/µL
MultiScribe
™
Reverse
Transcriptase (50 U/µL)
0.25 1.25 U/µL
Total
6.15
c
c. If changing the reaction volume, make sure the final proportions are consistent with the
recommended values above.
Note RT volume can vary from 10 µL to 100 µL. Increasing the RT volume
will reduce the total number of reactions.
—
Thermal Cycling Parameters for RT Reactions
Step
Incubation
a
a. If using random hexamers or oligo d(T)
16
primers for first-strand cDNA synthesis, a
primer incubation step (25 °C for 10 min) is necessary to maximize primer–RNA
template binding.
RT
Reverse
Transcriptase
Inactivation
HOLD HOLD HOLD
Time 10 min 30 min 5 min
Temperature 25 °C48°C95°C
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