Omega plasmid extraction process introduction

Experimental procedure

1. Take 1.5-5 ml of bacterial solution at 10,000 xg for 1 min at room temperature to collect the bacterial cell pellet;

2. Carefully remove the supernatant, add 250ul of Solution I/RNase, vortex or use a pipette to blow up and resuspend the cells.

3. Add 250ul of Solution II and mix gently by inverting 4-6 times to obtain a clear lysate.

4. Add 125ul Buffer N3, mix it upside down until a white flocculent precipitates, and let it react for 2-3 minutes at room temperature.

5. 12,000 × g at room temperature or 4 ° C for 10 min to obtain a supernatant, transfer the supernatant to a new centrifuge tube;

6. Add equal volume of ETR Binding Buffer to the supernatant, mix by inversion 7-10 times, and let stand for 5 min at room temperature.

7. Binding the plasmid - insert the binding column into a 2 ml collection tube, transfer 700 ul of the mixture to the binding column each time, centrifuge at 8,000 x g for 1 min, and discard the filtrate.

8. Repeat step 7 until all supernatants have been filtered and the filtrate is discarded.

9. Add 500 ul of ETR Wash Buffer to the binding column and centrifuge at 8,000 xg for 1 min to filter all of the liquid through the column and discard the filtrate.

10. Add 500ul Buffer EHB to the binding column, centrifuge at 8,000 × g for 1 min, filter all the liquid through the column, and discard the filtrate;

11. Add 700ul of DNA Wash Buffer to the binding column, centrifuge at 8,000 × g for 1 min to filter all the liquid through the column and discard the filtrate;

Note: Concentrated DNA Wash Buffer must be diluted with absolute ethanol as indicated on the label before use.

12. Add 700ul of DNA Wash Buffer to the binding column, centrifuge at 8,000 × g for 1 min to filter all the liquid through the column, and discard the filtrate;

13. Put the empty column back into the centrifuge tube and centrifuge the tube for 2 minutes at the maximum speed (not more than 1,300 × × ×);

14. Place the binding column in a new 1.5 ml centrifuge tube, add 30-50 ul Endotoxin-Free Elution Buffer, place at room temperature for 2-5 min, and centrifuge the plasmid DNA for 1 min at maximum speed.

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