Preparing sequencing samples:
- Combine the following in a 0.2ml PCR tube:
250-500ng DNA/sample,
2.2pmol primer/sample,
add PCR water to total volume of 8ul. - Add 4ul per sample of Master Mix (see recipe below).
Big Dye v3.1: 2 ul
Big Dye dilution buffer 2 ul - Run the sample in a ThermalCycler using the BigDye protocol as follows:
Step 1 Incubate at 96.0C for 20 seconds
Step 2 Incubate at 50.0C for 20 seconds
Step 3 Incubate at 60.0C for 4 minutes
Step 4 Repeat to Step 1 for 39 times
Step 5 Incubate at 12.0C forever
Step 6 End - Clean up the reaction with various methods.
Quantity of template DNA varies depending on the size of fragment.
Template size:
| Quantity | |||
| 100-200bp | 15-30ng | ||
| 500bp | 30-65ng | ||
| 1000bp | 65-130ng | ||
| 2000bp | 125-250ng | ||
| 3000bp | 250-500ng |
Big Dye dilution buffer:
| Tris-HCl,pH8.8 | 400mM | ||
| MgCl2 | 10mM |
Protocol adapted from http://www.osa.sunysb.edu/dna/faq.html.