TABLE 2.
Oligonucleotide primers
| Primer | Sequencea | Nucleotide positionb (range) | Annealing tempc (°C) |
|---|---|---|---|
| T3 (universal) | 5′-GCGCGCAATTAACCCTCACTAAAG-3′ | 53.0 | |
| T7 (universal) | 5′-GTAATACGACTCACTATAGGGC-3′ | 48.5 | |
| pBK13a-L1 | 5′-GCAACTAATGATAAAGAAGGC-3′ | 349-368 | 48.8 |
| pBK13a-R1 | 5′-GTAATGCCGCACTTTGCAGC-3′ | 3220-3229 | 51.8 |
| 13aMut-L | 5′-GAAAAATGgGAAGCAAATACATCA-3 | 1760-1783 | 51.3 |
| 13aMut-R | 5′-TGCTTCcCATTTTTCAATATTTTTTA-3 | 1774-1749 | 51.4 |
| 13aEcoR1-L | 5′-GCgaattcTGATGATAAGAACGAAAATTCAC-3′ | 1082-1112 | 49.5d |
| 13aNot1-R | 5′-AAgcggccgcACCCAGTGTCTTTTGATTTAAC-3′ | 2001-1970 | 49.2d |
| MagaP13a-L1 | 5′-AAACGGGGCTAAAGAAGCTG-3′ | 828-847 | 51.0 |
| MagaP13a-R1 | 5′-AAGCTGGTTATATTTTCCATATC-3′ | 2151-2173 | 48.9 |
| MagaP13a-L2 | 5′-TGTCAAAAATACAAATCTAGGTG-3′ | 1168-1190 | 48.8 |
| MagaP13a-R2 | 5′-CTTTAACTTGTGATGAGGTATC-3′ | 1823-1844 | 47.9 |
| MagaP13a-L3 | 5′-CGATATATGTAATTAGTGCTCTC-3′ | 47.9 | |
| MagaP13a-R3 | 5′-GTTCTTATCATCACATTTAGCAG-3′ | 48.6 |
a
Lowercase letters indicate nucleotides added to create restriction enzyme recognition sites and mutations for cloning.
b
Based on nucleotide sequence AJ315329.
c
Obtained with the PCR primer annealing temperature calculator developed by J. Boxall at http://www.iacr.bbsrc.ac.uk/res/depts/biochem/old-or-to-move/tcalculator.html by using the parameters 30% as the target GC content and 1,000 bp as the target size.
d
Nucleotides added to create the restriction enzyme recognition sites were not considered.