Reaction set-up |
|
| Component | Volume |
|---|---|
| Big Dye terminator premix: | 16 µl |
| ThermoFidelase: | 1 µl |
| primer: | 13 pmoles |
| DNA template: | 2-3 µg |
| di H20: | q.s. |
| total volume: | 40 µl |
Cycling conditions |
|
| 95° | 5 minute initial hold |
| 95° | 30 seconds |
| 55° | 20 seconds |
| 60° | 4 minutes |
45 cycles |
|
| 4° | hold |
Purification
Centri-Sep spin column
ABI 377 Gel/loading
resuspend in 2 µl and load all on a 48 cm 5.25% PagePlus gel with a 36 well comb
Templates Successfully Sequenced |
|
| Ureaplasma urealyticum | 0.75 Mb genome |
| Mycoplasma fermentans | 1.2 Mb genome |
| Streptococcus pneumoniae | 2.3 Mb genome |
| Escherichia coli | 4.6 Mb genome |