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Gene replacements in the tir-cesT-eae region of enterohemorrhagic E. coli 0157:H7 using long homology-containing substrates |
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| Exp # |
Substrate preparation |
Amount of DNA |
Genotype of linear fragment |
Total Transformants |
Percent transformants Amp sensitive |
Recombinants verified by PCR (# verified/# tested) |
|
|
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| Plasmid digests: |
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| 1. |
restriction digest pKM184 (SacI, SphI) |
2 μg |
Δeae::cat |
>500 |
11% (4/35) |
1/1 |
| 2. |
restriction digest pKM185 (SacI, SphI) |
2 μg |
Δeae-cesT-tir::cat |
103 |
2.6% (2/76) |
1/1 |
| 3. |
restriction digest pKM 187 (SacI, SphI) |
1 μg |
Δeae::cat sacB |
> 500 |
2.8% (1/35) |
1/1 |
| Plasmid digests including backbone: |
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| 4. |
restriction digest pKM184 (SacI, SphI, ApaLI) |
0.5 μg |
Δeae::cat |
52 |
57% (4/7) |
NT |
| 5. |
restriction digest pKM184 (SacI, SphI, ApaLI) |
10 μg |
Δeae::cat |
> 500 |
95% (143/151) |
10/10 |
| 6. |
restriction digest pKM185 (SacI, SphI, ApaLI) |
2 μg |
Δeae-cesT-tir::cat |
30 |
56% (9/16) |
9/10 |
| 7. |
restriction digest pKM185 (SacI, SphI, ApaLI) |
2 μg |
Δeae-cesT-tir::cat |
36 |
50% (9/18) |
7/9 |
| Gel-purified fragments: |
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| 8. |
purified SacI-SphI fragment from pKM185 |
0.5 μg |
Δeae-cesT-tir::cat |
225 |
100% (20/20) |
1/1 |
| 9. |
purified KpnI fragment from pKC2 |
0.1 μg |
Δtir::cat |
6 |
NT |
6/6 |
| 10. |
purified SacI-SphI fragment from pKM188 |
0.6 μg |
Δeae cesT tir::cat sacB |
273 |
100% (40/40) |
1/1 |
| 11. |
purified KpnI fragment from pKC3 |
0.05 μg |
Δtir::cat sacB |
2 |
NT |
2/2 |
Murphy and Campellone BMC Molecular Biology 2003 4:11 doi:10.1186/1471-2199-4-11 |
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