Excision of Clones as pBluescript from lZAPII¶

Contributed by Gordon W. Laurie, School of Medicine, University of Virginia, United States

Excision of Clones as pBluescript from lZAPII

Streak XLI-Blue MRF’ from frozen stock on an LB-tetracycline plate.
Incubate plate overnight at 37°C. Streak SOLR strain from frozen stock on an LB-kanamycin plate.
Pick a single XLI-Blue MRF’ colony and a single SOLR colony with a sterile toothpicks to each inoculate 10 ml of LB broth in 50 ml conicals.
Grow overnight at 30°C.
Warm water bath to 70°C
Add 50 ml of LB to 0.5 ml of each overnight culture, and grow at 37°C for 2-3 hours in a 250 flask.
Check OD600 at 2 hours and every 30 min thereafter.
Wish to have XLI-Blue MRF’ in mid-log phase at OD600 of 0.2-0.5. Allow SOLR to grow to OD600 of 0.5-1.0; before they reach OD600 ? 1.0, remove from incubator and let sit at RT on rotator.
Spin XLI-Blue MRF’ for 10 minutes at 2,766 rpm in a sterile 50 ml conical using Beckman GS 6R centrifuge.
Carefully decant media and gently resuspend pellet at OD600 = 1.0 in 10 mM MgSO4 (3-4 ml).
For each clone, in a 50 ml conical, combine:

Incubate at 37°C for 15 minutes.
Add 3 ml of LB broth and incubate for 2 hours at 37°C with shaking.
Spin down cells for 15 minutes at 3,194 rpm in Beckman GS 6R centrifuge.
Transfer supernatant to a Sartstedt 55.514 tube.
Heat tube at 70°C for 15 minutes.
Spin in a Sorvall centrifuge with a SA600 rotor at 5,262 rpm for 15 minutes.
Decant the phage supernatant into a sterile tube. Add 200 µl of OD600 ? 1.0 SOLR cells into two 1.5 ml eppendorf tubes (2 tubes/clone); add 100 µl of supernatant to one tube and 10 µl to the other tube.
Incubate at 37°C for 15 minutes.
Plate 10 µl, 50 µl and 10 µl of 1/10 - 1/1000 diluted onto LB amp plates using a ethanol flamed glass spreader and rotator. Plate SOLR cells alone as a negative control.

This method is based, with permission, on an original protocol available here.