Prepare LB agar plates containing:
| Final
concentration
|
Stock solution concentration |
| 50 µg/ml kanamycin | 10 mg/ml in water |
| 7 µg/ml gentamicin | 7 mg/ml in water |
| 10 µg/ml tetracycline | 10 mg/ml in ethanol |
| 100 µg/ml Blue-gal | 20 mg/ml in DMSO |
| 40 µg/ml IPTG | 200 mg/ml in water |
1.Thaw the DH10Bac competent cells on ice.
2. Add 1 µl of DNA into 40 µl competent DH10Bac. Place the mix into 0.2 mm cuvette on ice.
3. Program the electroporator to 200 ohm
25 µF
2.5 Kvolts
4. Place the cuvette in the electroporator and push both buttons till beep.
5. Add 0.9 ml warm SOC medium to cuvette and take out to snap-cap tube.
6. Place the mixture in a shaking incubator at 37oC for 4 h.
7. Serially dilute the cells using SOC medium to 10-1, 10-2, 10-3 (100 µl of transposition mix + 900 µl of SOC medium = 10-1 dilution, use this to further dilute 10-fold to give 10-2 dilution, and similarly to 10-3 dilution).
11. Place 100 µl of each dilution on the plates and spread evenly over the surface.
12. Incubate for 48 h at 37oC (colonies are very small and blue colonies may not be discernible prior to 24 h).
13. Pick large white colonies for colony
PCR (see procedure)