Protocol taken from Stratagene's Gigapack packaging extracts instruction manual
Host bacteria (E. coli LE392)preparation:
- Pick one colony from fresh overnight culture on NZY agar plate and inoculate 50 ml NZY broth supplemented with 0.2% maltose and 10 mM MgSO4.
- Grow at 37C, shaking, 4-6 h (do not grow past OD600 1.0/ml).
- Pellet bacteria 2000 rpm for 10 min.
- Resuspend cells in one-half original volume sterile 10 mM MgSO4.
- Dilute cells to OD600 of 0.5 with sterile 10 mM MgSO4. Plating bacteria should be no older than 48 h.
DNA packaging:
- Remove extracts from freezer and place on dry ice. Start thawing sonic extract (yellow tube) first.
- Quickly thaw freeze-thaw extract (red tube) until just beginning to thaw.
- Add DNA immediately (1-4 ml containing 0.1-5 mg) to freeze-thaw extract. Place on ice.
- Quickly add 15 ml sonic extract to DNA-freeze-thaw extract.
- Stir or pipet to mix, avoiding introduction of air bubbles.
- Incubate at room temperature for 2 h.
- Add 500 ml phage dilution (SM) buffer. Store at 4 C. Supernatant is ready to be titered.
Transfection: titering library
- Make 1/10 and 1/50 dilutions of packaged DNA in SM buffer.
- Mix 25 ml of undiluted and diluted samples with 25 ml cells as prepared above. Let stand at room temperature for 30 min.
- Add 200 ml LB and incubate 1 h at 37 C, mixing gently every 15 min.
- Spin tubes for 1 min and resuspend pellet in 50-100 ml LB, and spread on L-agar plates containing selective antibiotic.
Amplifying cosmid library
- In a series of small culture tubes or microfuge tubes, mix packaged DNA with equal volume of host cells. Incubate 30 min at room temperature.
- Add 4 volumes LB, incubate 1 h at 37 C, with shaking. Resuspend and plate as above. Volumes to be plated depends on titer of packaged DNA and size of plates used.
- To store library, pour 3 ml LB onto plate and scrape colonies into the broth with sterile razor blade. Resuspend cells, pool, add appropriate antibiotic to cell suspension, and freeze aliquots at -80 C.
