CosmidCloning:Cellpreparation,DNApackaging,andCellTransfection

Cosmid Cloning: Cell preparation, DNA packaging, and Cell Transfection

Protocol taken from Stratagene's Gigapack packaging extracts instruction manual

Host bacteria (E. coli LE392)preparation:

1. 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.

2. Grow at 37C, shaking, 4-6 h (do not grow past OD600 1.0/ml).

3. Pellet bacteria 2000 rpm for 10 min.

4. Resuspend cells in one-half original volume sterile 10 mM MgSO4.

5. Dilute cells to OD600 of 0.5 with sterile 10 mM MgSO4. Plating bacteria should be no older than 48 h.

DNA packaging:

1. Remove extracts from freezer and place on dry ice. Start thawing sonic extract (yellow tube) first.

2. Quickly thaw freeze-thaw extract (red tube) until just beginning to thaw.

3. Add DNA immediately (1-4 ml containing 0.1-5 mg) to freeze-thaw extract. Place on ice.

4. Quickly add 15 ml sonic extract to DNA-freeze-thaw extract.

5. Stir or pipet to mix, avoiding introduction of air bubbles.

6. Incubate at room temperature for 2 h.

7. Add 500 ml phage dilution (SM) buffer. Store at 4 C. Supernatant is ready to be titered.

Transfection: titering library

1. Make 1/10 and 1/50 dilutions of packaged DNA in SM buffer.

2. Mix 25 ml of undiluted and diluted samples with 25 ml cells as prepared above. Let stand at room temperature for 30 min.

3. Add 200 ml LB and incubate 1 h at 37 C, mixing gently every 15 min.

4. 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

1. 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.

2. 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.

3. 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.