Table 1 PCR reactions used to map intron insertions in DFCT3, DFCT9, DFCT13, and DFCT16
Reaction | Primers | Target | Notes |
|---|---|---|---|
1 | V1F–V1R | rsbV1 | Confirms intron insertion (primers flank insertion site) |
2 | incAF–incAR | incA | Confirms intron insertion (primers flank insertion site) |
3 | hyp08F–hyp08R | cryptic plasmid | Confirms that transformation has not resulted in loss of the native, cryptic plasmid |
4 | incAF–GIIR | Connects the 5′ region of incA to the intron | Confirms intron insertion and orientation |
5 | incAR–GIIF | Connects the 3′ region of incA to the intron | Confirms intron insertion and orientation |
6 | GIIF–GIIR | Intron | Confirms intron presence |
7 | incAF–aadAR | Connects incA to the aadA marker | Confirms marker type |
8 | catF–catR | Intron donor plasmid | Confirms loss of intron donor plasmid |
9 | V1F–GIIF | Connects the 5′ region of rsbV1 to the intron | Confirms intron insertion and orientation |
10 | V1R2–GIIR | Connects the 3′ region of rsbV1 to the intron | Confirms intron insertion and orientation |
11 | blaR2–VIR | Connects rsbV1 to the bla marker | Confirms marker type |
12 | blaF1–blaR1 | Marker | Confirms presence of bla marker |
13 | aadAF–aadAR | Marker | Confirms presence of aadA marker |