Team:LCG-UNAM-Mexico/Resources/Protocols/Purification protocol

Purification of T7 from 35 ml lysates
A standard stock of each new recombinant can be prepared by using a single plaque to infect 35 ml of growing cells in a shaking 125 ml flask (or other convenient volume, such as 50 ml in a 250 ml flask). This volume of lysate can be processed in a single 50 ml polypropylene centrifuge tube so that many lysates can be processed at the same time.

1. Inoculate 50 ml of sterile LB broth in a 250 ml Erlenmeyer flask with a single colony of an appropriate T7 host strain (e.g., BL21 for T7Select415 vectors, BLT5403 or BLT5615 for T7Select1 and T7Select10-3 vectors). Shake at 250 rpm overnight at 37°C. If using the BLT5403 or BLT5615 host strain, include 50 μg/ml carbenicillin in the media.

2. The next morning, add 0.15 ml of the fresh overnight culture to 35 ml M9LB or M9TB. Shake at 37°C until the OD600 reaches 0.6–0.8 (about 3.5 h). If using BLT5615, add IPTG to 1 mM when the OD600 reaches 0.4 and continue shaking for another 30 min.

3. Using a large-bore pipet tip, transfer a plug containing a single T7 plaque from an agar plate, or add 5 μl of high titer phage lysate to the culture. Continue shaking at 37°C until lysis (usually 1–1.5 h). Lysis will be recognized by “clearing” of the culture; strings of cell debris and a translucent appearance instead of the cloudy appearance of intact cells.

4. Upon lysis, add 3 ml of 5 M NaCl to 35 ml cultures (or 5 ml of 5 M NaCl to the 50 ml cultures, swirl to mix, and transfer to 50 ml Falcon tubes. Centrifuge at 7,000 rpm in a Beckman JA-12 rotor or equivalent for 10 min at 4°C.

5. To prepare phage stocks, transfer 5 ml of the supernatant to a sterile tube (e.g., 15 ml Falcon). If frozen stocks are desired, add 1 ml supernatant to 0.1 ml sterile 80% glycerol in a cryovial, mix thoroughly, and store at -80°C. Store the remainder tightly capped at 4°C as the working lysate.

6. Pour the remaining supernatant from step 4 into a fresh 50 ml Falcon tube. Add 1/6 volume of sterile 50% PEG 8000 (100 g PEG dissolved with stirring in 100 ml deionized water and autoclaved, and then brought to 200 ml with sterile deionized water). Cap the tube and mix thoroughly by vortexing. Thorough mixing is important because the 50% PEG is very viscous, and if a small film were to remain at the bottom of the tube, the high concentration of PEG would prevent subsequent extraction of phage from the pellet.

7. Allow the lysate-PEG mixture to stand on ice for at least 30 min to precipitate the phage particles. The mixture can be left indefinitely at this stage, and it is often convenient to leave tubes overnight in the refrigerator.

8. Centrifuge the lysate-PEG mixture for 10 min at 7,000 rpm and carefully decant the supernatant. Allow excess liquid to drain away from the pellet by leaving the tubes in an inverted position on paper towels for a few min.

9. Add 1.2 ml of 1 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA to the drained pellet and vortex vigorously for 30 seconds to extract the phage.

10. At this point, transfer the mixture to a 1.5 ml microcentrifuge tube. Centrifuge at 12,000 × g for 10 min and layer the supernatant on a CsCl step gradient in an SW50.1 ultracentrifuge tube prepared as described above.

11. Centrifuge 35 min at 40,000 rpm at room temperature to band the phage. Collect the phage as described above. If collecting with a pipet from above, first remove the top layer of debris to within approximately 1 cm of the phage band. Use a fresh Pasteur pipet to collect the phage band in as small a volume as possible. Transfer the phage into a sterile polypropylene tube, cap tightly, and store at 4°C. Typically, about 0.2 ml of purified phage at a concentration of a few times 1012 per ml is obtained, although the concentration may be lower for recombinants that grow poorly.