fUSE13CT4.doc 03/01/06 Page 2

Midipreps of RF from fd-tet-based display vectors

This procedure assumes that cells harboring an fd-tet-based display phage are available, either as colonies on a plate or in some other form. These phage bear the inducible tetracycline resistance determinant of Tn10. Previously we used CsCl density equilibrium centrifugation in the presence of a saturating concentration of ethidum bromide to purify covalently closed circular RF from other contaminants. More recently, however, we have used ion exchange chromatography on QIAGEN columns for this purpose in order to avoid working with large amounts of the potentially carcinogenic ethidium bromide. Although the RF preparation obtained by ion exchange chromatography is not nearly as pure as that from CsCl/ethidum bromide density equilibrium centrifugation, it seems to be adequately pure for library construction and other applications. A 6-liter procedure (easily down-sized by a factor of 6) is illustrated in RFmidiprep2.doc. The current midiprep protocol is on the scale of 200 ml.

PROCEDURE

1. Use a single well-separated colony to inoculate 200 ml NZY plus 0.2 µg/ml tetracycline[1] in a 1-liter culture flask; shake vigorously for 2 hr at 37º; add additional tetracycline to 20 µg/ml; continue to shake vigorously overnight at 37º.

2. Pour the culture into a 250-ml centrifuge bottle; centrifuge 10 min 4º 5 Krpm in SLA1500 rotor; RRR; resuspend cells in 39 ml TE + 1 ml 5 M NaCl; pour and pipette into a 50-ml tube; centrifuge 5 Krpm 10 min 4º in FiberLite rotor[2]; RRR. (If necessary, cell pellet can be frozen at this point; however, if possible just go straight on to the next step.)

3. Thoroughly resuspend the pellet in the 50-ml tube in 9 ml QIAGEN buffer P1.

4. Add 9 ml QIAGEN buffer P2 (large QIAGEN box labeled QIAGEN Plasmid Midi Kit (25)). Mix gently but thoroughly by inverting 6 times (don’t vortex!!), and incubate at room temperature for 5 min (no more).

5. Add 9 ml pre-chilled QIAGEN buffer P3; mix immediately but gently by inverting 6 times (don’t vortex); incubate on ice 30 min.

6. Centrifuge at 14 Krpm 30 min at 4º in FiberLite rotor; pour supernatant through cheesecloth into a fresh 50-ml tube.

7. Centrifuge at 14 Krpm 15 min at 4º in FiberLite rotor; carefully pour supernatant into a fresh 50-ml tube.

8. To the tube (total volume nominally 27 ml) add 19 ml isopropanol at room temperature; mix thoroughly by ~50 inversions; mark one edge of the tube to designate the centrifugal wall (where the pellet will end up); orienting the marked edge outward (centrifugally), centrifuge at 14 Krpm at 4º for 30 min in FiberLite rotor; RRR (be very careful and gentle as glassy pellet may not be visible); air dry briefly.

9. Add 500 µl TE, pumping and scraping the centrifugal wall at the bottom to completely redissolve the pelleted DNA.

10. Add 4.5 ml QIAGEN buffer QBT; vortex thoroughly to mix; centrifuge the tube briefly in the clinical centrifuge to drive the solution to the bottom.

11. Mount a QIAGEN-tip 100 over a waste container; equilibrate the tip with 4 ml buffer QBT, allowing the effluent to drain to the waste. The column will stop flowing automatically when the meniscus reaches the upper frit.

12. Still collecting to waste, apply the midiprep in the 50-ml tube to the column and allow the entire sample to flow through.

13. Still collecting to waste, wash the column twice with 10 ml QIAGEN buffer QC, allowing the entire wash buffer to flow through each time.

14. Position the QIAGEN-tip column to collect into a fresh 50-ml tube. Elute the column with 5 ml QIAGEN buffer QF; again, allow the buffer to flow completely through the column.

15. To the 50-ml tube (with 5 ml eluate) add 3.5 ml isopropanol; mix thoroughly by vortexing; mark one edge of the tube to designate the centrifugal wall (where the pellet will end up); orienting the marked edge outward (centrifugally), centrifuge at 14 Krpm at 4º for 30 min in FiberLite rotor; RRR (be very careful and gentle as glassy pellet may not be visible).

16. Gently pipette 3 ml freezer-cold 70% ethanol down the centripetal edge (away from marked centrifugal edge); roll the tube gently to allow the wash to gently pass over the pellet; again orienting the marked edge outward (centrifugally), re-centrifuge at 14 Krpm at 4º for 10 min in FiberLite rotor; RRR (again be very careful and gentle as glassy pellet may not be visible); air dry 15 min.

17. Dissolve the pellet in 300 µl 1/10 × TE by pumping and scraping the centrifugal wall (where the pellet, possibly invisible, should lie); centrifuge the tube briefly in the clinical centrifuge to drive solution to the bottom; transfer the solution to 500-µl Ep tube; after removing a 5-µl sample next step, store it in the refrigerator or deepfreeze.

18. Scan 100 µl of a 1/20 dilution (diluent & reference = 1/10 × TE) from 220 to 300 nm; a typical nucleic acid concentration is 200–800 µg/ml; only a fraction of the nucleic acid is actual RF.

[1] This concentration of tetracycline is sub-inhibitory but sufficient to induce expression of the tetA gene (the resistance protein). The 2-hr induction period before challenge with an inhibitory concentration of tetracycline ensures that a clone will be propagated even if it’s phenotypically sensitive at the time of inoculation. Usually this precaution isn’t necessary, since TetA mediated resistance is phenotypically unusually stable.

[2] These excellent rotors allow you to spin standard disposable 50-ml conical screw-cap centrifuge tubes at 15,000 rpm! The pellet collects at the angle rather than the tip, which makes it particularly easy to free of residual supernatant. If you don’t have this rotor, you can use OakRidge tubes and the Sorvall SS34 rotor (or equivalent Beckman rotor) instead.