BigDye Labeling Reaction Conditions
| Template DNA* | *Either 200 ng plasmid or 10-20 ng PCR product for every 200 bp sequence |
|---|---|
| Primer | 10pmol |
| Big Dye Terminator V3.1 | 1 ul |
| 5x Sequencing Buffer | 3 ul |
| Total Volume | 10 ul |
| Standard Thermal Cycling Conditions | ||
|---|---|---|
| 96° C | 5min. | 1 cycle |
| 96° C | 15 sec. | 25 cycles |
| 50° C | 15 sec. | |
| 60° C | 4 min. | |
| 4° C | hold | |
| Difficult Template Thermal Cycling Conditions** | ||
|---|---|---|
| 98° C | 10 min. | 1 cycle |
| 98° C | 30 sec. | 30 cycles |
| 50° C | 30 sec. | |
| 60° C | 4 min. | |
| 4° C | hold | |
** For difficult to sequencing templates (GC rich or hairpins), DMSO must be added at 5% final concentration to the BigDye labeling reaction.
Send comments or questions regarding this page to the UR Genomics Research Center
