Escherichia coli , Pseudomonas putida and Bacillus subtilis . As you can see, amino acid residue at the C-terminus of UP1 region is not conserved so much. Additional sense primers, UP1E and UP1G have been designed to match this diversity. In contrast, the UP2r region does not display as much diversity. An antisense primer, UP2Ar was designed to match this genotype.
Sequence is as follows.
3' TAC ATR CTR CGN CTR CAN CTR CCG AGC GTG TAG GCA TGG GAC GA 5' UP2rS 3' CCG AGC GTG TAG GCA TGG GAC GA 5' Troubleshooting primers With the primers listed above, gyrB fragments can be amplified for the vast majority of bacteria. However, we still experienced difficulty amplifying gyrB in certain strains. Because the amplified gyrB and parE fragments are not separated by agarose gel electrophoresis and it is therefore difficult to determine the nucleotide sequence without any cloning steps. To overcome this, we designed gyrB specific primers from GyrB and ParE sequence alignments. In some strains of the genus Streptomyces , there are two copies of gyrB in their genome.