Table 1 Conditions and primers for hiTAIL-PCR
Reaction | Numbers of cycles | Thermal settings |
|---|---|---|
Primary | 1 | 93°C 1 min, 95°C 1 min |
5 | 94°C 1 min, 60°C 1 min, 72°C 1.5 min | |
1 | 94°C 1 min, 25°C 3 min, ramping to 72°C in 3 min | |
15 | 94°C 30 s, 63°C 1 min, 72°C 2.5 min | |
94°C 30 s, 63°C 1 min, 72°C 2.5 min | ||
94°C 30 s, 44°C 1 min, 72°C 2.5 min | ||
1 | 72°C 8 min, 4°C forever | |
Secondary | 12 | 94°C 30 s, 63°C 1 min, 72°C 2.5 min |
94°C 30 s, 63°C 1 min, 72°C 2.5 min | ||
94°C 30 s, 44°C 1 min, 72°C 2.5 min | ||
1 | 72°C 8 min, 4°C forever | |
Tertiary | 20 | 94°C 30 s, 63°C 1 min, 72°C 2.5 min |
94°C 30 s, 63°C 1 min, 72°C 2.5 min | ||
94°C 30 s, 44°C 1 min, 72°C 2.5 min | ||
1 | 72°C 8 min, 4°C forever | |
Primers | Size (bp) | Sequencesa |
AD1 | 15 | 5' NTCGASTWTSGWGTT3' |
AD2 | 16 | 5' NGTCGASWGANAWGAA3' |
AD3 | 16 | 5' WGTGNAGWANCANAGA3' |
dSP1 | 24 | 5' ATTGGGAACGACGCTGCCGTTATA 3' |
dSP2 | 24 | 5' ACGGAATACCCATGACGATTTTGG3' |
dSP3 | 24 | 5' CTGGCGTTGTGATTCGTGGTTTGT 3' |
uSP1 | 24 | 5' CGGCTTCCAAAATCGTCATGGGTA 3' |
uSP2 | 24 | 5' TATAACGGCAGCGTCGTTCCCAAT 3' |
uSP3 | 24 | 5' CAGCCGCGTACGTGAACAAAAATG 3' |
uSP4 | 24 | 5' CAGCTCCGGCCAATCTGAGGGTAA 3' |
uSP5 | 24 | 5' ATCAAGGGGATGCGCTCGCAGACA 3' |
uSP6 | 24 | 5' CGGAAACCTTTCACCTGCCAGCAA 3' |