Answer 6: Our standard induction is 15 minutes. However, we know that shorter and longer times can work depending on the details of the experiment. Below it can be seen that with oligo recombination, 5 minutes induction is sufficient for optimal recombination levels. However with dsDNA recombination, a 10 minute or longer induction time is required. Likewise, long inductions with the Beta-only strain, SIMD50, are ok. This table does not include the data but long induction times of HME6 (and most recombineering strains) results in cell killing if the induction is 60 minutes or longer due to the lambda kil gene.
| LENGTH OF INDUCTION (min) | SIMD50 | HME6 | |
|---|---|---|---|
| Gal+ /108 viable 1 | Gal+ /108 viable 1 | CatR /108 viable 2 | |
| 0 | 3.1x104 | 1.7x104 | 2.0x101 |
| 1 | 9.0x104 | 1.2x104 | |
| 3 | 7.5x106 | 1.2x106 | 1.4x102 |
| 5 | 2.0x107 | 1.5x107 | 2.1x104 |
| 10 | 3.2x107 | 3.1x107 | 1.2x105 |
| 15 | 1.6x107 | 2.8x107 | 1.3x105 |
| 20 | 1.2x107 | 2.6x107 | 6.4x104 |
| 30 | 1.2x107 | 1.6x107 | 1.1x104 |
| 60 | 1.1x107 | ||
| overnight (saturated) | <2x101 | ||
| Overnight then dilute at 42° | 6.2x106 | ||
1 Recombination with an oligo to correct the galKam to Gal+.
2 Recombination with a chloramphenicol cassette to knock-out galK.
© RECOMBINEERING 2016