From 2010.igem.org

Qiagen Midi-prep plasmid isolation

This protocol is based on QIAGEN® Plasmid Purification Handbook. This protocol is designed for preparation of up to 100 µg of high- or low-copy plasmid or cosmid DNA using the Qiagen Plasmid Midi Kit. Qiagen plasmid isolation is used to obtain very pure plasmid DNA.

Maximum recommended culture volumes for the Qiagen-tip 100:

High-copy plasmids 25 mL

Low-copy plasmids 100 mL

Materials:

- bacterial culture

- Qiagen colums

- buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0)

- buffer P2 (200 mM NaOH, 1% SDS)

- buffer P3 (3 M KAc, pH 5.5)

- buffer QBT (750 mM NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0, 0.15% Triton X-100)

- buffer QC (1 M NaCl, 50 mM MOPS, 15% Ethanol, pH 7.0)

- buffer QF (1.25 M NaCl, 50 mM Tris/HCl, 15% Ethanol, pH 8.5)

- isopropanol

- milliQ

- centrifuge

- nanodrop

Protocol:

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 25 to 100 mL LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm)

2. Harvest the 25 to 100 mL bacterial cells by centrifugation at 4.000 rpm for 15 min at 4°C ( Sorvall buckets). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C

3. Resuspend the bacterial pellet in 4 mL of chilled Buffer P1 (containing RNAse) and pour into a blue cap 50 mL tube

4. Add 4 mL of Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 minutes

5. Add 4 mL of chilled Buffer P3, mix gently by inverting 4–6 times, and incubate on ice for 15 minutes

6. Centrifuge 30 min at 4.000 rpm and pour supernatant through cheesecloth into a fresh blue cap 50 mL tube

7. During centrifugation equilibrate a Qiagen-tip 100 by applying 4 mL Buffer QBT, and allow the column to empty by gravity flow

8. Apply the supernatant from step 6 onto the Qiagen-tip and allow it to enter the resin by gravity flow

9. Wash the Qiagen-tip with 2 × 10 mL Buffer QC

10. Elute DNA with 5 mL Buffer QF into 15 mL blue cap tube

11. Precipitate DNA by adding 3.5 mL (0.7 volumes) room-temperature isopropanol to the eluted DNA

12. Mix and centrifuge immediately at 4.000 rpm for 30 minutes at 4 °C

13. Proceed immediately when centrifuge stops and carefully decant the supernatant

14. Wash DNA pellet with 2 mL of cold 70% ethanol, and centrifuge at 4.000 rpm for 10 minutes at 4 °C

15. Carefully decant the supernatant without disturbing the pellet

16. Air-dry the pellet for 5–10 min (has to be dry), and redissolve the DNA in a suitable volume of milliQ H2O

17. Measure DNA concentration on the Nanodrop.