Table 2 Primers and PCR conditions used for deletion and confirmation of various genes in S. Typhimurium LT2 (NCBI accession no. NC_003197.2).
Serial No | Primer Name | Sequence | Binding location of primer in genome of S. Typhimurium LT2 | Amplicon size | Purpose | PCR conditions | References |
|---|---|---|---|---|---|---|---|
1 | MsrA deletion Test Forward | 5′ AGATACATTAATGTTGTTATT 3′ | 4,645,918 to 4,645,938 | 300 bp in Δ5msr and 850 bp in S. Typhimurium | Confirmation of msrA deletion | 95 °C 5 min 95 °C 30 s 48 °C 40 s × 35 72 °C 90 s 72 °C 10 min | |
2 | MsrA deletion Test Reverse | 5′ GTAACGTTTAATGAAAACCG 3′ | 4,645,089 to 4,645,108 | ||||
3 | MsrB deletion Forward | 5′AGTTGGTACTGAGGTGTTAATGTTTTGTTAGAATCGGTCAGGCAATGTAGGCTG 3′ | – | 1096 bp | To amplify FRT flanked Chloramphenicol cassette | 95 °C 5 min 95 °C 30 s 42 °C 30 s × 35 72 °C 90 s 72 °C 10 min | Current study |
4 | MsrB deletion Reverse | 5′CATGATGTGTCCCCTCCTGTGGAATAATTTGCTGAATCGTTTTTTATGAATA 3′ | – | ||||
5 | MsrB deletion Test Forward | 5′ ATTCGCCTCACTCTTCCTTTCG 3′ | 1,369,809 to 1,369,830 | 498 bp in Δ5msr and 869 bp in S. Typhimurium | Confirmation of msrB deletion | 95 °C 5 min 95 °C 30 s 62 °C 40 s × 40 72 °C 100 s 72 °C 10 min | Current study |
6 | MsrB deletion Test Reverse | 5′ CCATCTGACCGTGAGTATCGA 3′ | 1,370,657 to 1,370,677 | ||||
7 | MsrC deletion Test Forward | 5′ GAGCAAGAACGCATTTAATGC 3′ | 1,944,530 to 1,944,550 | 280 bp in Δ5msr and 500 bp in S. Typhimurium | Confirmation of msrC deletion | 95 °C 5 min 95 °C 30 s 48 °C 40 s × 40 72 °C 90 s 72 °C 10 min | |
8 | MsrC deletion Test Reverse | 5′ GCGTACGCAGGCCGTGTT 3′ | 1,944,054 to 1,944,071 | ||||
9 | MsrP deletion Forward | 5′GACCGGGAGTCTGTGATGAAAAAGATACGTCCATTAACAGAAGCCGTGTAGGCTGGAGCTGCTTC 3′ | – | 1092 bp | To amplify FRT flanked Chloramphenicol cassette | 95 °C 5 min 95 °C 30 s 62 °C 40 s × 35 72 °C 100 s 72 °C 10 min | |
10 | MsrP deletion Reverse | 5′TGCTGTCAGACGCACTTAAAAATTCTCCCGCAAATTGAGACCGCGCATATGAATATCCTCCTTAG 3′ | – | ||||
11 | MsrP ST test Forward | 5′AGGGCCGTACGCTGGTGAAGAT 3′ | 3,547,964 to 3,547,985 | 550 bp in Δ5msr and 1401 bp in S. Typhimurium | Confirmation of msrP deletion | 95 °C 5 min 95 °C 30 s 62 °C 40 s × 40 72 °C 100 s 72 °C 10 min | |
12 | MsrP ST test Reverse | 5′GAAACACCATAATCCTAACAGGCG 3′ | 3,549,341 to 3,549,364 | ||||
13 | BisC deletion Forward | 5′CTCCCTGCAAACCGTTGTGCATGACCAGGTGCACAGTAAAACGCGGTAGGTGG 3′ | – | 1103 bp | To amplify FRT flanked Chloramphenicol cassette | 95 °C 5 min 95 °C 30 s 42 °C 30 s × 35 72 °C 90 s 72 °C 10 min | Current study |
4 | BisC deletion Reverse | 5′CGCCTGCGGTAGGTTCAGGGTCCGGCCATGCGCCTTCGTGAATACAATGAATAT 3′ | – | ||||
15 | BisC ST test Forward | 5′TTGACCCACGCACCATCGCGT 3′ | 3,832,645 to 3,832,665 | 445 bp in Δ5msr and 2335 bp in S. Typhimurium | To amplify FRT flanked Chloramphenicol cassette | 95 °C 5 min 95 °C 30 s 64 °C 40 s × 40 72 °C 150 s 72 °C 10 min | Current study |
16 | BisC ST test Reverse | 5′TTATGAGTTGGCAGGCGGATCAA 3′ | 3,830,332 to 3,830,354 |
