MIDI PREP by alkaline lysis/Qiagen-tip 100 isolation

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This protocol yields ~100 µg plasmid DNA and meets the needs of most large-scale plasmid uses in this lab.

You will need:
- buffers STE, P1, P2, P3, QBT, QC, QF
- one Qiagen-tip 100
- two clean, autoclaved 250 ml centrifuge bottles
- three clean, autoclaved 50 ml centrifuge bottles

for pBluescript derivatives (Tac/Lac family)

1. innoculate 3 ml LB (+ appropriate antibiotic) with a fresh O/N colony; grow to mid-log phase, 37 C, shaking

2. innoculate 100 ml warm LB (+ antibiotic) with ~3 ml culture from 1.; grow 14-16 hrs., 37 C, shaking

3. transfer culture to two 250 ml centrifuge bottles; spin @ 4000 rpm for 15 min., 4 C.

4. pour off SN completely

5. wash (suspend) culture in 20 mls ice-cold STE and transfer to two 50 ml centifuge bottles; spin @ 4000 rpm for 15 min., 4 C.

6. pour off STE completely

7. suspend cell pellet in 4 ml buffer P1 (pellets may be combined, but be sure to counterbalance your sample properly)

8. add 4 ml buffer P2, mix, and leave RT for 5 min. 9. add 2 ml chilled buffer P3, mix, and chill on ice for 15 min.

10. spin down cell lysate @ 16,000 rpm for 30 min., 4 C

11. near the end of this centrifuge step, equilibrate a Qiagen-tip 100 by allowing ~4 ml QBT to flow through it

12. as soon as this centrifuge step (step 10.) is finished, apply the SN to Qiagen-tip 100; allow to flow through

13. wash Qiagen-tip 100 with 2x 10 ml buffer QC

14. elute DNA with 5 ml buffer QF into a clean 50 ml centrifuge bottle, ppt DNA with 3.5 ml isopropanol

15. spin down DNA @ 11,000 rpm for 30 min. 4 C

16. pour off SN; wash DNA pellet with 5 ml cold 70% EtOH, air dry 5 min.; suspend in appropriate buffer/volume

Buffers

STE 100 mM NaCl 10 mM Tris 1 mM EDTA adjust to pH 8.0 (w/ HCl) QBT 750 mM NaCl 50 mM MOPS adjust to pH 7.0 15% EtOH 0.15% Triton X-100 P1 (store 4 C) 50 mM Tris 10 mM EDTA 100 µg/ml RNase A adjust to pH 8.0 (w/ HCl)

QC 1.0 M NaCl 50 mM MOPS adjust to pH 7.0 15% EtOH P2 200 mM NaOH 1% SDS QF 1.25 M NaCl 50 mM Tris adjust to pH 8.5 (w/ HCl) 15% EtOH P3 (store 4 C) 3.0 M KOAc adjust to pH 5.5 (w/ glacial acetic)

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Made Feb. 22, 1999; Baker Laboratory

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