Table 2.
Primers and resulting fragments used in overlap extension PCR for construction of PrtH + and PrtH + WANS mutants
| Fragment | Primer | Sequence (5′ – 3′) | Length (bp) | DNA matrix (strain) | Product size (kbp) | Hyb temp (°C) | Overlap product size (kbp) | Overlap hyb temp (°C) |
|---|---|---|---|---|---|---|---|---|
| PrtH+ |
|
|
|
|
|
|
|
|
| F1 |
Style 1-1* |
ACAAATTCATGCCGTTCATAAG |
22 |
gDNA (LMD-9) |
0.986 |
59.5 |
8.622 |
55 |
| PrtSssUp(H)_R |
GCCTTAACTTGTTGTTCTGCAGCTACCGATGGTGC |
35 |
||||||
| F2 |
PrtHpp_F |
GAACAACAAGTTAAGGCTAGTGTTGACAGCCAAACAAAAAC |
41 |
gDNA (CNRZ32 CIRM-BIA 103) |
5.752 |
62 |
||
| PrtHterm_R |
AAAAGAGTAATGATCCTTCTCATTACTCTTTCATTATATGTAAATGATTATTTTAC |
56 |
||||||
| F3 |
EmpG9(H)_F |
GAGAAGGATCATTACTCTTTTCAAACTTAAGAGTGTGTTGATAGTGC |
47 |
pG+host 9 |
0.969 |
59.5 |
||
| EmpG9_R |
GGACCTCTTTAGCTCCTTGG |
20 |
||||||
| F4 |
PrtSDn(Em)_F |
CCAAGGAGCTAAAGAGGTCCATAATAAAACCGCTTAATCATTGTG |
45 |
gDNA (LMD-9) |
0.973 |
49 |
||
| PrtSDn_R |
CGTCTATCAATCTTGTATTTTCTTG |
25 |
||||||
| PrtH+WANS |
|
|
|
|
|
|
|
|
| F5 |
UpH_F |
CGGTATCAAGTGGGGTACTCG |
21 |
gDNA (PrtH+) |
1.395 |
69 |
4.186 | 55 |
| UpH(WANS)_R |
CTTGCTTGGCTTGCAGAAACAGGAGCTGCAACTTGGTTATC |
41 |
||||||
| F6 |
WANS_F |
TCTGCAAGCCAAGCAAG |
17 |
gDNA (LMD-9) |
0.473 |
58 |
||
| WANS(aphA3)_R |
CTCAAATGGTTCGCTGGTTAGCAAACTTGTGATAAAGC |
38 |
||||||
| F7 |
aphA3_F** |
CCAGCGAACCATTTGAG |
17 |
gDNA (TIL1193) |
1.4 |
54 |
||
| aphA3_R** |
GTTGCGGATGTACTTCAG |
18 |
||||||
| F4 |
PrtSDn(aphA3)_F |
CTGAAGTACATCCGCAACATAATAAAACCGCTTAATCATTGTG |
43 |
gDNA (LMD-9) |
0.971 |
61.7 |
||
| PrtSDn_R | CGTCTATCAATCTTGTATTTTCTTG | 25 | ||||||
Primers Style 1-1, PrtSDn_R, and UpH_F correspond to those used for overlapping PCR reaction and italicised nucleotides correspond to homologous tails.
The name, sequence, length, DNA matrix, product size (including the added tails), hybridization temperature (hyb temp), overlap product size and overlap hybridisation temperature (hyb temp) are indicated.
bp (base pairs); kbp (kilobase pairs) and gDNA (genomic DNA) * [42], ** [18].