1. Inoculate 5 ml of 2xTY containing the appropriate antibiotic in glass tube (18 cm height 16 mm diameter) with 5 µl of the glycerol stock.
2. Grow for 18h at 37 °C with linear shaking (tubes should be in an almost horizontal position).
3. Transfer the bacteria to 10 ml centrifuge tubes.
4. Pellet bacteria at 2000g for 10 minutes.
5. Pour off the supernatant.
6. Add 550 µl of P1 and resuspend the pellet.
7. Transfer to 96-square well block (2ml/well Polyfiltronics).
8. Add 550 µl of P2. Close the block with a self adhesive pad and mix by inversion (10 times) and leave for 5 minutes at RT.
9. Add 550 µl of P3. Close the block with a self adhesive pad, mix by inversion (10 times) and leave on ice for 30 minutes with occasional shaking.
10. Spin at 6200g for 45 minutes.
11. Transfert 1.2 ml (or more) of supernatant from each well to another 96-square well block.
12. Shear the DNA by passing the supernatant seven times through a needle (21G X 1'').
13. Setup the QIAwell system (96 columns).
14. Load the sheared supernatant onto the columns and apply vacuum until the columns run dry (- 35Kpa).
15. Wash the column with 1 ml of QW (twice).
16. Elute the DNA with 250 µl of QF heated to 70 °C (twice).
17. Add 2 µl of glycogen (2 mg/ml) (Appligene).
18. Add 350 µl of isopropanol and mix.
19. Spin at 6000g for 1 hour and pour off the supernatant.
20. Add 500 µl of EtOH 70%.
21. Spin at 6000g for 15 minutes and pour off the supernatant.
22. Dry the pellet (speed vac).
23. Add 30 µl of TE and leave overnight at RT.
24. Transfer into a 96-well microtiter plate and store at -20 C.

Notes

The buffers used in this protocol P1, P2, P3, QW, QF are from QIAGEN.

We have established automated equipment for simultaneous processing of 96 samples for steps 11 and 12.

We are currently establishing automated equipment for simultaneous processing of 96 samples for steps 1, 2 and 3.

If using this protocol please refer to :
M. Salanoubat and F. Quetier

Genoscope - Centre National de Sequençage EVRY FRANCE.