Protocol BigDye v.3.1
Hovedinnhold
1. Quantifying DNA
| Template | Quantity |
| PCR product: | |
| 100-200 bp | 1-3 ng |
| 200-500 bp | 3-10 ng |
| 500-1000 bp | 5-20 ng |
| 1000-2000 bp | 10-40 ng |
| >2000 bp | 20-50 ng |
| Single-stranded | 25-50 ng |
| Double-stranded | 150-300 ng |
| Cosmid, BAC | 0,5-1.0 µg |
| Bacterial genomic DNA | 2-3 µg |
2. Preparing sequencing reaction
| Reagent | Quantity |
| Big-Dye version 3.1 | 1 µl |
| Sequencing buffer (store at 4C) | 1 µl |
| Template (see table above) | 200 ng |
| Primer | 3,2 pmol |
| Deionised water q.s.p. 10 µl | |
| Total 10 µl |
N.B. Alternatively, use up to 1,5 µl of the sequencing buffer and 5 pmol of primer
3. Performing cycle sequencing in a thermal cycler
1 96º C, 5 min
2 25 cycles 96 º C, 10 sec.
50 º C, 5 sec. *
60 º C, 4 min.
3 4 º C
* Annealing temp should be optimized according to the primer used (typicallY Tm-5)
N.B. Add 10 µl deionised water after the sequencing reaction to deliver your sample
13.01.2015