Table 1.
Primers and PCR Protocol Information
| primer pair |
primer sequencesa (5′ to 3′) | product (length in base pairs) | annealing temperature, extension time |
|---|---|---|---|
| 1 | GAGTAGGCACTCCGCACCATGTCACTAACATCGTTTTACGCCC + CTACAAGCTTCGACTAGGCCACCCACACC | cloA with easA promoter extension (2491 bp) | 63 °C, 75 s |
| 2 | GACCTCTAGACATTGCTTCTAATCCACCAAGTACTTG + GGCGTAAAACGATGTTAGTGACATGGTGCGGAGTGCCTACTC | easA promoter with cloA extension (814 bp) | 64 °C, 30 s |
| 3 | GACCTCTAGACATTGCTTCTAATCCACCAAGTACTTG + CTACAAGCTTCGACTAGGCCACCCACACC | easA promoter-cloA fusion product (3253 bp) | 63 °C, 100 s |
| 4 | GAGTAGGCACTCCGCACCATGTCACTAACATCGTTTTACGCCC + CCGCCGATGTCATACTTCAACTAGTATGTGACTTCAGTCCATC | processed cloA with easA promoter and 3′ UTR extensions (1562 bp) | 63 °C, 40 s |
| 5 | GACTGAAGTCACATACTAGTTGAAGTATGACATCGGCG + CACAGAATTCGAACTTCTGTGACGTCGCCAGATTG | 3′ UTR with cloA extension (422 bp) | 63 °C, 20 s |
| 6 | GACCTCTAGACATTGCTTCTAATCCACCAAGTACTTG + CACAGAATTCGAACTTCTGTGACGTCGCCAGATTG | easA promoter-processed cloA-3′ UTR fusion product (2713 bp) | 64 °C, 90 s |
| 7 | GTACCAGACGAATCTACACAATGTCACTAACATCGTTTTACGCC + CCGCCGATGTCATACTTCAACTAGTATGTGACTTCAGTCCATC | processed cloA with gpdA promoter and 3′ UTR extensions (1564 bp) | 61 °C, 40 s |
| 8 | GATTCTAGAAGTCCTGAATAGTAG + GGCGTAAAACGATGTTAGTGACATTGTGTAGATTCGTCTGGTAC | gpdA promoter with cloA extension (990 bp) | 64 °C, 40 s |
| 9 | GATTCTAGAAGTCCTGAATAGTAG + CACAGAATTCGAACTTCTGTGACGTCGCCAGATTG | processed cloA with gpdA promoter and 3′ UTR fusion product (2889 bp) | 60 °C, 90 s |
| 10 | CTATAGAGTAGGCACTCCGCAC + CTACGCCGACTACTGTTGTCC | cloA cDNA (1524−1621 bp) | 58 °C, 45 s |
| 11 | CAGTTCGAGCACGTCTTCTCG + GTGCTCGACAATCATGAGGTC | lpsA near frame shift (743 bp) | 60 °C, 30 s |
a
Underlines indicate unique restriction sites used for cloning fusion PCR products: TCTAGA, XbaI; GAATTC, EcoRI; and AAGCTT, HindIII.