Team:KULeuven/Protocols

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• Miniprep (Qiagen)
• ...

Plasmid DNA purification

Materials

• LB broth with appropriate antibiotic
• 15 ml tube
• incubation oven at 37°C
• microcentifuge tubes
• table-top microcentrifuge
• buffer P1 (Qiagen kit)
• buffer P2 (Qiagen kit)
• buffer N3 (Qiagen kit)
• QIAprep spin columns
• transparant tape
• buffer PE (Qiagen kit)
• buffer EB (Qiagen kit)
• nanodrop

Procedure

1. Inoculate a single colony into 5 ml of LB with the appropriate antibiotic and incubate at 37°C for 12-16 hours (liquid culture).
2. Put 1.5 ml of this liquid culture in a microcentrifuge tube and centrifuge at 8500 rpm for 3 minutes at room temperature.
3. Remove all traces of supernatant by inverting the tube.
4. Resuspend the pelleted cells in 250 $\mu$l Buffer P1 and vortex until no cell clumps remain.
5. Add 250 ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times. !! Do not vortex and do not allow the reaction to proceed for more than 5 minutes !
6. Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. The solution will become cloudy.
7. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will form.
8. Apply the supernatant of step 4 to a QIAprep spin column by decanting. When you label the columns, cover this label with tape (the ink tends to dissolve).
9. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through. If you made several tubes of one sample, you can repeat the steps 8 and 9 in the same QIAprep spin column. This way, the concentration of plasmid DNA will be higher.
10. Wash the QIAprep spin column with 0.75 ml Buffer PE. Centrifuge for 1 minute at 13000 rpm. Discard the flow-through !!
11. Centrifuge for an additional minute to remove all residual wash buffer.
12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 ul Buffer EB to the centre of the QIAprep spin column, let stand for 1 minute and centrifuge for 1 minute at 13000 rpm.
13. Measure the concentration of plasmid DNA with the nanodrop (ng/ul). Use Buffer EB as blank.

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