Table 2 Published primers and nested RT-PCR conditions
Primer sequences | ||
|---|---|---|
1st PCR | ||
Forward | 5′-GGN TGG GAY TAY CCN AAR TGY GA-3′ | 760 bp amplicon |
Reverse | 5′-RHG GRT ANG CRT CWA TDG C-3′ | |
2nd PCR | ||
Forward | 5′-GGT TGG GAC TAT CCT AAG TGT GA-3′ | 440 bp amplicon |
Reverse | 5′-CCA TCA TCA GAT AGA ATC ATC AT-3′ | |
Reverse transcription & First PCR | ||||
|---|---|---|---|---|
Master mix constituents per reaction | PCR profile | |||
5× RT buffer | 5 µL | |||
dNTP (10 mM) | 1 µL | 45 °C | 30 m | |
5× Q solution | 5 µL | 95 °C | 15 m | |
Forward primer (200 µM) | 0.5 µL | 94 °C | 30 s | |
Reverse primer (200 µM) | 0.5 µL | 45 °C | 30 s | 50× |
One-step enzyme mix | 1 µL | 72 °C | 1 m | |
RNase-free water | 2 µL | 72 °C | 10 m | |
RNA | 10 µL | 15 °C | Hold | |
Total | 25 µL | |||
Second PCR | ||||
|---|---|---|---|---|
Master mix constituents per reaction | PCR profile | |||
10x PCR buffer | 5 µL | |||
MgCl2 (25 mM) | 3 µL | 95 °C | 10 m | |
dNTP (10 mM) | 1 µL | 95 °C | 15 s | |
Forward primer (10 µM) | 1 µL | 45 °C | 30 s | 35× |
Reverse primer (10 µM) | 1 µL | 72 °C | 30 s | |
PCR-grade water | 33.5 µL | 72 °C | 5 m | |
AmpliTaq Gold™ | 0.5 µL | 15 °C | Hold | |
Purified 1st PCR amplicon | 5 µL | |||
Total | 50 µL | |||