Sequencing Ustilago maydis with the
LI-COR 4200 DNA Sequencer

Dr. Dagmar Schuette, Sequencing Department
LION Bioscience AG, Heidelberg, Germany

Methods

Plasmid DNA Preparation:
Bacterial colonies were transferred to 96-deep well blocks containing 1.3 ml 2YT medium for 21 hours (37°C; 250 rpm, 20° angle). DNA was prepared using a Qiagen bioRobot 9600. The DNA content was measured in a 96-well UV photometer (Molecular Devices) and brought to a concentration of 100 ng/µL.

High Throughput Plasmid DNA Sequencing:
For standard reactions, both sides of the inserts were sequenced simultaneously by adding two different labeled oligonucleotides (Doublex Sequencing; Wiemann et al. 1995) and standard dye-primer sequencing biochemistry (PE Biosystems). The reagents were combined on a Canberra Multiprobe II EX System (Packard). The reaction mixtures were incubated for 30 cycles (15'' at 95°; 20'' at 50°C; 30'' at 68°C) in ABI 9700 thermocyclers (PE Biosystems).

Afterwards, probes were dried and resolved in denaturation buffer. The denaturated sequencing products were loaded onto paper combs (Erfle et al. 1997) and separated on LI-COR 4200 sequencers. The performance was strictly controlled by the LION Laboratory Information Management System (LIMS).