Please note: These include new primer sequences (added 26 July 2004; labelling corrected on 5 March, 2007) whose labels indicate the genomic direction rather than reading frame.
The primer pairs for the PCR amplification of internal fragments of these genes can be chosen from:
583 bp 54° C
fumCR1 5'-TCCCGGCAGATAAGCTGTGG-3'
fumCF 5'-TCACAGGTCGCCAGCGCTTC-3'
fumCR 5'-GTACGCAGCGAAAAAGATTC-3'
806 bp 54° C
gyrBF 5'-TCGGCGACACGGATGACGGC-3'
gyrBR1 5'-GTCCATGTAGGCGTTCAGGG-3'
gyrBR 5'-ATCAGGCCTTCACGCGCATC-3'
911 bp 60° C
icdF 5'-ATGGAAAGTAAAGTAGTTGTTCCGGCACA-3'
icdR 5'-GGACGCAGCAGGATCTGTT-3'
878 bp 54° C
mdhF 5'-ATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGG-3'
mdhR 5'-TTAACGAACTCCTGCCCCAGAGCGATATCTTTCTT-3'
mdhF1 5'-AGCGCGTTCTGTTCAAATGC-3'
mdhR1 5'-CAGGTTCAGAACTCTCTCTGT-3'
932 bp 60° C
purAF1 5'-TCGGTAACGGTGTTGTGCTG-3'
purAF 5'-CGCGCTGATGAAAGAGATGA-3'
purAR 5'-CATACGGTAAGCCACGCAGA-3'
816 bp 54° C
recAR1 5'-AGCGTGAAGGTAAAACCTGTG-3'
recAF 5'-CGCATTCGCTTTACCCTGACC-3'
recAF1 5'-ACCTTTGTAGCTGTACCACG-3'
recAR 5'-TCGTCGAAATCTACGGACCGGA-3' 780 bp 58° C
Conditions:
PCR: 2 min at 95°, 30 cycles of 1 min at 95°, 1 min at annealing temp, 2 min at 72° followed by 5 min at 72°. The PCR reaction contains 50 ng of chromosomal DNA, 20 pmol of each primer, 200 umol (10 ul of a 2 mM solution) of the dNPTs, 10 ul of 10x PCR buffer, 5 units of Taq polymerase and water to 100 ul.
Sequencing
We use the amplification primer pairs for the sequencing step.