Sequencing Reaction Protocol
Sequencing reactions are performed by PCR using the following conditions:
| BDV3.1 | 1µl |
| 5x Dilution Buffer | 5µl |
| Primer (5µM) | 1µl |
| Deionized H2O | make up to a final volume of 20µl |
| DNA (100ng/kb) | should not exceed 10µl |
- For BAC DNA add 4µl of BDv3.1 and 2µl of Dilution Buffer
- For GC rich templates add DMSO/glycerol to a final 5% (v/v) concentration
Please note that when determining how much DNA to add to a reaction, the entire plasmid size should be taken into account and not just the insert.
If sequencing a PCR product, the PCR product needs to be purified. We recommend using Shrimp Alkaline Phosphatase and ExonucleaseI (please contact us for product information).
Cycling Conditions:
| Temp. | Time | Cycles |
|---|---|---|
| 94oC | 5min | x1 cycle |
| 94oC | 10sec | x30 cycles |
| 50oC | 5sec | |
| 60oC | 3mins |
- When sequencing BAC DNA, optimal product concentration is critical. Amplify at [(95°C -5min) x1, (95°C -30sec; 50°C -10sec; 60°C -4min) for at least 50 cycles].
- When sequencing with primers that have a high Tm (>65°C), eliminate the annealing step.
- When dealing with high GC rich templates, increase the initial denaturation to 98°C for 10min, followed by 98°C for 30sec; 50°C-5sec; 60°C-3min for 30 cycles.
The most common problem we see with samples is the failure to remove unincorporated dyes from the sequencing reaction. AGD highly recommend plate and spin column purification (please contact us for product information).