Team:UEA-JIC Norwich/miniprep

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1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue.

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless.

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.

6. Centrifuge for 30–60 s. Discard the flow-through.

7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details)

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.

9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

10. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.