Team:TU Delft/protocols/midi-prep plasmid isolation


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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

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 or 20 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 buffer (e.g., TE buffer pH 8.0). Normally 100 mL TE buffer or autoclaved milliQ H2O was used.

17. Measure DNA concentration on the Nanodrop

Used buffers:

Buffer P1: 100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0

Buffer P2: 200 mM NaOH, 1% SDS (Make fresh, every two weeks)

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

TE buffer: 10 mM Tris/HCl, 0.1 mM EDTA, pH 8.0