Team:UQ-Australia/Notebook/Miniprep procedure



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Miniprep Procedure
Production of Cleared Lysate

1. Pellet 1-10ml overnight cultures for 5 minutes

2. Thoroughly re-suspend pellet with 250ul Cell Resuspension Solution

3. Add 250ul Cell Lysis Solution to each sample; invert 4 times to mix

4. Add 10ul Alkaline Protease Solution; invert 4 times to mix. Incubate 5 minutes at room temperature

5. Add 350 ul Neutralizer Solution; invert 4 times to mix

6. Centrifuge at top speed for 10 minutes at room temperature.

Binding of Plasmid DNA

7. Insert Spin Column into Collection Tube

8. Decant cleared lysate into Spin Column

9. Centrifuge at top speed for 1 minute at room temperature. Discard flowthrough and reinsert Column into Collection Tube.

Washing

10. Add 750ul Wash Solution (ethanol added). Centrifugeat top speed for 1 minute. Discard flowthrough and reinsert column into Collection Tube

11. Repeat step 10 with 250ul Wash Solution

12. Centrifuge at top speed for 2 minutes at room temperature

Elution

13. Transfer Spin Column to a sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column.

14. Add 100ul of Nuclease-free Water to the Spin Column. Centrifuge at top speed for 1 minute at room temperature.

15. Discard column and store DNA at -20'C or below.