Note: Orientation A and B refer to the conventions used in Ellermeier et al (2002, in press, Gene). Each set of primers is designed to precisely replace the open reading frame with the template insertion (starting at P1/P4 and ending at P4/P1). These primers are all designed with 40-nt homology regions, and 20 nt annealing. The screening primers (5'pcr and 3'pcr) are homologous to the 20 nt directly flanking the 40-nt homology regions on each end of the deletion. They have not been tested for quality in any way.

Note: I've posted a protocol for doing this technique in Salmonella on our protocols page.

• Salmonella enterica serovar Typhimurium LT2 (gb:AE006468) + pSLT

  • Orientation A P1/P4 primers
    This set of primers is oriented such that P1 is at the 5' end of the gene. I use this orientation for my knockouts.
    
  • Orientation B P1/P4 primers
    This set of primers is oriented such that P4 is at the 5' end of the gene.
    
  
  

• Salmonella enterica serovar Typhi CT18 (gb:AL513382) + pHCM1 + pHCM2

  • Orientation A
    Note that the first genes from each plasmid are not included in the primer list.
    
  
  

• Escherichia coli K-12 (gb:U00096)

  • Orientation A
    
  
  

• Escherichia coli O157:H7 (gb:BA000007)

  • Orientation A
    
  
  

• Escherichia coli O157:H7 EDL933 (gb:AE005174)

  • Orientation A
    Note that this sequence has many unresolved nucleotides ("N"), which prevented primers from being designed for approximately 6 genes.
    
  
  

• Yersinia pseudotuberculosis IP 32953

  • NC_006153 Orientation A
  • NC_006154 Orientation A
  • NC_006155 Orientation A
  
  

• Yersinia pestis CO92

  • NC_003131 Orientation A
  • NC_003132 Orientation A
  • NC_003134 Orientation A
  • NC_003143 Orientation A
  
  

• Yersinia pestis KIM

  • NC_004088 Orientation A
  
  

• Yersinia pestis biovar Mediaevails str. 91001

  • NC_005810 Orientation A
  • NC_005813 Orientation A
  • NC_005814 Orientation A
  • NC_005815 Orientation A
  • NC_005816 Orientation A