Utah State/18 June 2008

Mini-Scale preparation for DNA Sequencing by CTAB-DNA Precipitation STET buffer: 8% sucrose, 50 mM EDTA, 0.1% TritonX-100, 50 mM Tris-HCl (pH 8)
 * Grow cells overnight in LB with 50 micrograms/ml Ampicillin
 * Recover cells by centrifugation in a microcentrifuge tube for 20 seconds. For one DNA labeling reaction, 1.5 ml of cell culture is needed.
 * Resuspend pellet in 200 microliters of STET buffer.
 * Add 4 microliters of Lysozyme (50 mg/ml) and incubate at room temperature for 5 min.
 * Boil for 45 seconds and centrifuge for 20 minutes.
 * Remove pellet using a toothpick.
 * Add 5 microliters of RNase A (10 mg/ml) and incubate at 68 C for 10 minutes.
 * Add 10 microliters 5% CTAB and incubate at room temperature for 3 minutes.
 * Centrifuge for 5 minutes and resuspend in 300 microliters 1.2 M NaCl by vortex mixing.
 * Add 750 microliters ethanol and centrifuge for 5 minutes.
 * Rinse pellet in 80% ethanol, dry, and resuspend in TE.