UCL London/Protocol/Miniprep

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Miniprep

Materials:

• Miniprep Kit: P1, P2, N3, PB, PE, EB buffer
• pin column tubes

Method:

1. Label Set 1 eppendorfs; Label Spin column tubes; Label Set 2 eppendorfs.
2. Add ~1.5ml E.coli suspension in Set 1 eppendorfs; and then centrifuge for 3min at 8000rpm & discard all the supernatant.
3. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
4. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
5. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
6. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
7. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
8. Centrifuge for 30–60 s. Discard the flow-through.
9. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) # Store at -20°C.