Fluorescent-labeled sequencing gel preparation, pre-electrophoresis, sample loading, electrophoresis, data collection, and analysis on the ABI 373A DNA sequencer

ID: 82

Group:

Methods for DNA sequencing

Prepared by:

Bruce A. Roe (broe@uoknor.edu)

Detail:

Polyacrylamide gels for fluorescent DNA sequencing are prepared as described above except that the gel mix is filtered prior to polymerization. Optically-ground, low fluorescence glass plates are carefully cleaned with hot water, distilled water, and ethanol to remove potential fluorescent contaminants prior to taping. Denaturing 6% polyacrylamide gels are poured into 0.3 mm X 89 cm X 52 cm taped plates and fitted with 36 well forming combs. After polymerization, the tape and the comb are removed from the gel and the outer surfaces of the glass plates are cleaned with hot water, and rinsed with distilled water and ethanol. The gel is assembled into an ABI sequencer, and the checked by laser-scanning. If baseline alterations are observed on the ABI-associated Macintosh computer display, the plates are recleaned. Subsequently, the buffer wells are attached, electrophoresis buffer is added, and the gel is pre-electrophoresed for 10-30 minutes at 30 W.

Year prepared: NULL

Date uploaded: 2010-02-26 14:28:18

url:

Laboratory Protocols