UCL London/Protocol/Miniprep

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.