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Star Republic: Guide for Biologists

Midi/Maxi plasmid prep column (Qiagen)

1. Qiagen plasmid midi prep can be used to purify up to 100 mg of plasmid or cosmid DNA. Qiagen plasmid maxi prep can be used to purify up to 500 mg of plasmid or cosmid DNA. Purified DNA usually is good enough for transfection and micro-injection.

2. Grow 25-100 ml of bacteria culture for one midi prep and 100-500 ml for maxi prep.

3. Chill P3 on ice.

4. Harvest bacterial cells by centrifugation at 6,000 g for 15 minutes at 4 C.

5. Discard supernatant, and resuspend the bacterial pellet in 4 ml (midi) or 10 ml (maxi) of P1/RNAse by repeated pipetting up and down.

6. Add 4 ml (midi) or 10 ml (maxi) of P2, mix by inverting 4-6 times or by pipetting up and down, and incubate at room temperature for 5 minutes.

7. Add 4 ml (midi) or 10 ml (maxi) of P3, mix by inverting 4-6 times or by pipetting up and down. Incubate on ice for 15-20 minutes.

8. Centrifuge at >20,000 g for 30 minutes at 4 C. Remove supernatant to a fresh tube promptly.

9. Re-centrifuge the supernatant at >20,000 g for 15 minutes at 4 C. Remove supernatant to a fresh tube promptly.

10. Equilibrate a QIAGEN-tip 100 (midi) or QIAGEN-tip 500 (maxi) by applying 4 ml (midi) or 10 ml (maxi) Buffer QBT, and allow the column to empty by gravity flow.

11. Add the supernatant containing DNA to the QIAGEN-tip and allow it to empty by gravity flow.

12. Wash the QIAGEN-tip with 10 ml (midi) or 30 ml (maxi) Buffer QC.

13. Repeat step 12.

14. Elute DNA with 5 ml (midi) or 15 ml (maxi) Buffer QF. Collect with fresh Corex centrifugation tubes.

15. Precipitate DNA by adding 0.7 volumes isopropanol. Mix and centrifuge at 15,000 g for 30 minutes at 4 C. Discard supernatant.

16. Wash DNA pellet with 2-10 ml 70% ethanol. Carefully decant the supernatant without disturbing the pellet.

17. Allow the pellet to air dry. Resuspend the DNA in 100-500 ml of TE.