TABLE 1.
Primers and PCR parameters for analysis of DNA templates prepared by the boiling method
| Primera | Target size (kb) | Primer sequence (5′-3′) | PCR cycling parameters |
|---|---|---|---|
| PCR1 | |||
| JL99 | 3.1 | TTT CAC GGG TTG GGG TTT CTA CAG G | 95°C for 1 min, 62°C for 1 min, 72°C for 5 min |
| JL100 | 3.1 | GAC GGG GAA CCT CTT TAC AAA CAT C | |
| PCR2 | |||
| JL102 | 2.1 | CGC AAG GTG AGG GTT CGA ACC GAG G | 95°C for 1 min, 68°C for 1 min, 72°C for 3 min |
| JL103 | 2.1 | CCG CAG CAG GGA GGC AAA CAA TGA A | |
| PCR3 | |||
| JL221 | 3.5 | CCA GAA AAT GCG AAG GTA AGT GCT TC | 95°C for 1 min, 55°C for 1 min, 72°C for 5 min |
| JL222 | 3.5 | GCG ACA CGG AAA TGT TGA ATA CTC AT | |
| Control | |||
| JL136 | 1.4 | AGG CTC GAA AGG CGC ATA CGA | 95°C for 1 min, 68°C for 1 min, 72°C for 2 min |
| JL137 | 1.4 | GTG GTC TAC TGC CCA GCC ATC |
a
PCR1 primers are for integration into the 5′ region of nor-1 (pAPGUSNP, pBNG3.0), PCR2 primers are for integration into the 3′ region of nor-1 (pAPGUSNP, pBNG3.0), PCR3 primers are for integration into pyrG (pAPGUSNP), and control primers are for endogenous vbs exon II (pAPGUSNP).