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Summary of the diverse conditions used and final protocol |
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| Stepa |
Condition |
Obtention of recombinant colonies (CFU/ml)b |
Final established condition |
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|
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| Construction of the amplimer |
Short homologous arms |
No |
Long primers |
| Long homologous arms |
Yes |
Single PCR reaction for tet |
|
| Single PCR reaction |
21 for Tc |
Three PCR reaction for cat |
|
| Three PCR reactions |
19 for Cm |
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| Amount of amplified DNA to be transformed |
0.1 μg |
0 |
0.5 μg |
| 0.25 μg |
0 |
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| 0.5 μg |
21 Tc, 19 Cm |
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| Culture volume used for preparation of electrocompetent cells |
5 ml (2 × 109 CFU/ml) |
0 |
50 ml (5.1010 CFU/ml) |
| 10 ml (1010 CFU/ml) |
0 |
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| 25 ml (2.5 × 1010 CFU/ml) |
1 Tc, 0 Cm |
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| 50 ml (5 × 1010 CFU/ml) |
9 Tc, 8 Cm |
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| Temperature after electroporation |
30°C |
2 Tc, 1 Cm |
37°C |
| 37°C |
15 Tc, 9 Cm |
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| Arabinose concentration media |
1 mM |
18 Tc, 9 Cm |
0.1 M |
| 10 mM |
10 Tc, 8 Cm |
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| 0.1 M |
20 Tc, 11 Cm |
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| Concentration of antibiotic in plating media |
Tc 20 μg/ml, Cm 20 μg/ml |
1 Tc, 0 Cm |
Tc 5 μg/ml, Cm 5 μg/ml |
| Tc 5 μg/ml, Cm 5 μg/ml |
21 Tc, 19 Cm |
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a Each set of experiments was performed independently of the other tests with one phage for each antibiotic. b Phage used for tet was ØVTB55; Phage used for cat was ØA9. | |||
Serra-Moreno et al. BMC Molecular Biology 2006 7:31 doi:10.1186/1471-2199-7-31 |
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