Part:BBa_V1003:Experience

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Applications of BBa_V1003

http://www.genome.bnl.gov/Vectors/pSCANScookbook.html

Preparation of High Efficiency Electrocompetent D1210 Cells.

Preparation of cells Inoculate 500 ml of 2XYT media (containing Streptomycin 30 µg/ml) with 1/100th volume of fresh overnight culture. Grow cells at 37°C with vigorous shaking to an ABS600 of 0.5 to 0.7. equal to about 3-4 x108 cfu/ml To harvest, chill the flask on ice for 15 minutes and centrifuge in a cold rotor at 4,000 x g max for 15 minutes. Remove as much of the supernatant as possible. Resuspend pellets in a total of 500 ml of ice-cold sterile ddH20. Centrifuge as in step 3. Resuspend cells in a total of 250 ml of ice-cold sterile ddH20. Centrifuge as in step 3. Resuspend cells in 20 ml of ice-cold sterile 10% glycerol. Centrifuge as in step 3. Resuspend cells in 1-2 ml in ice-cold sterile 10% glycerol. The cell concentration should be about 1-3 x 1010 cells/ml. Freeze the suspended cells in 50 ul aliquots on dry ice - ethanol and store at -80°C. The cells are good for at least 6 months under these conditions. Keep the cells as close to 0°C as possible throughout their preparation Preparation of pSCANS plasmid with or without insert.

E. coli D1210 carries a LacIq allele on the chromosome which ensures tight repression of the P1 replication origin in the pSCANS vector. Most LacIq strains carry this allele on incompatible F' plasmids, therefore they cannot be used as hosts for the F-based pSCANS vectors. D1210 is a derivative of the highly transformable strain HB101 [F- lac (i+o+z+y-) Gal- Pro- Leu- Thi- EndoI- hsm hsr recA rpsl] that also carries the lacy+ allele on the chromosome, which facilitates IPTG uptake leading to excellent, reproducible amplification of the pSCANS vector (with or without inserts) after addition of IPTG to the medium. Template preparation.

Bacterial colonies containing library plasmid are plated on 2XYT plus kanamycin plates. Colonies are picked and grown in 1 ml of 2XYT with shaking at 37° C without addition of IPTG for 5 hrs in 96 deep-well plates. An aliquot of each culture well is then mixed with an equal vol. of 20% glycerol and the samples are archived at -70° C in 96-well microtiter dishes in case a clone needs to be regrown at some future date. IPTG (1 mM final conc.) is added to the remaining portion of each culture well to induce plasmid amplification and incubation is continued overnight. High quality sequencing templates are prepared using an alkaline lysis protocol, followed by neutralization in the presence of a proprietary ion exchange resin (Edge BioSystems, Gaithersburg, MD) to bind cellular debris, which are then removed by filtration, and plasmid DNA is recovered by isopropanol precipitation. All steps in the procedure are performed in a 96-well format as are the resulting sequencing reactions.

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